CA2469738A1 - Expression vectors encoding epitopes of target-associated antigens and methods for their design - Google Patents

Abstract

The invention disclosed herein is directed to methods of identifying a polypeptide suitable for epitope liberation including, for example, the steps of identifying an epitope of interest; providing a substrate polypeptide sequence including the epitope, wherein the substrate polypeptide permits processing by a proteasome; contacting the substrate polypeptide with a composition including the proteasome, under conditions that support processing of the substrate polypeptide by the proteasome; and assaying for liberation of the epitope. The invention further relates to vectors including a housekeeping epitope expression cassette. The housekeeping epitope(s) can be derived from a target-associated antigen, and the housekeeping epitope can be liberatable, that is capable of liberation, from a translation product of the cassette by immunoproteasome processing. The invention also relates to a method of activating a T cell comprising contacting a substrate polypeptide with an APC
and contacting the APC with a T cell.

Description

EXPRESSION VECTORS ENCODING EPITOPES OF
TARGET-ASSOCIATED ANTIGENS AND METHODS FOR THEIR DESIGN
Background of the Invention Field of the Invention S [0001] The invention disclosed herein is directed to methods for the design of epitope-encoding vectors for use in compositions, including for example, pharmaceutical compositions capable of inducing an immune response in a subject to whom the compositions are administered.
The invention is further directed to the vectors themselves. The epitope(s) expressed using such vectors can stimulate a cellular immune response against a target cell displaying the epitope(s).
Description of the Related Art [0002] The immune system can be categorized into two discrete effector arms.
The first is innate immunity, which involves numerous cellular components and soluble factors that respond to all infectious challenges. The other is the adaptive immune response, which is customized to respond specifically to precise epitopes from infectious agents.
The adaptive immune response is further broken down into two effector arms known as the humoral and cellular immune systems. The humoral arm is centered on the production of antibodies by B-lymphocytes while the cellular arm involves the killer cell activity of cytotoxic T
Lymphocytes.

[0003] Cytotoxic T Lymphocytes (CTL) do not recognize epitopes on the infectious agents themselves. Rather, CTL detect fragments of antigens derived from infectious agents that are displayed on the surface of infected cells. As a result antigens are visible to CTL only after they have been processed by the infected cell and thus displayed on the surface of the cell.

[0004] The antigen processing and display system on the surface of cells has been well established. CTL recognize short peptide antigens, which are displayed on the surface in non-covalent association with class I major histocompatibility complex molecules (MHC). These class I peptides are in turn derived from the degradation of cytosolic proteins.
Summary of the Invention [0005] Embodiments of the invention provide expression cassettes, for example, for use in vaccine vectors, which encode one or more embedded housekeeping epitopes, and methods for designing and testing such expression cassettes. Housekeeping epitopes can be liberated from the translation product of such cassettes through proteolytic processing by the immunoproteasome of professional antigen presenting cells (pAPC). In one embodiment of the invention, sequences flanking the housekeeping epitope(s) can be altered to promote cleavage by the immunoproteasome at the desired location(s). Housekeeping epitopes, their uses, and identification are described in U.S. Patent Application Nos. 09/560,465 and 09/561,074 entitled EPITOPE

SYNCHRONIZATION IN ANTIGEN PRESENTING CELLS, and METHOD OF EPITOPE
DISCOVERY, respectively; both of which were filed on April 28, 2000.

[0006] Examples of housekeeping epitopes are disclosed in provisional U.S.
Patent Applications entitled EPITOPE SEQUENCES, Nos. 60/282,211, filed on April 6, 2001;
60/337,017, filed on November 7, 2001; 60/363210 filed 3/7/02; and 60/409,123, filed on September 5, 2002; and U.S. Application No. 10/117,937, filed on April 4, 2002, which is also entitled EPITOPE SEQUENCES.

[0007] In other embodiments of the invention, the housekeeping epitope(s) can be flanked by arbitrary sequences or by sequences incorporating residues known to be favored in immunoproteasome cleavage sites. As used herein the term "arbitrary sequences"
refers to sequences chosen without reference to the native sequence context of the epitope, their ability to promote processing, or immunological function. In further embodiments of the invention multiple epitopes can be arrayed head-to-tail. These arrays can be made up entirely of housekeeping epitopes. Likewise, the arrays can include alternating housekeeping and immune epitopes.
Alternatively, the arrays can include housekeeping epitopes flanked by immune epitopes, whether complete or distally truncated. Further, the arrays can be of any other similar arrangement. There is no restriction on placing a housekeeping epitope at the terminal positions of the array. The vectors can additionally contain authentic protein coding sequences or segments thereof containing epitope clusters as a source of immune epitopes. The term "authentic" refers to natural protein sequences.

[0008] Epitope clusters and their uses are described in U.S. Patent application Nos.
09/561,571 entitled EPITOPE CLUSTERS, filed on April 28, 2000; 10/005,905, entitled EPITOPE
SYNCHRONIZATION IN ANTIGEN PRESENTING CELLS, filed on November 7, 2001; and 10/026,066, filed on December 7, 2001, also entitled EPITOPE SYNCHRONIZATION
IN
ANTIGEN PRESENTING CELLS.

[0009] Embodiments of the invention can encompass screening the constructs to determine whether the housekeeping epitope is liberated. In constructs containing multiple housekeeping epitopes, embodiments can include screening to determine which epitopes are liberated. In a preferred embodiment, a vector containing an embedded epitope can be used to immunize HLA transgenic mice and the resultant CTL can be tested for their ability to recognize target cells presenting the mature epitope. In another embodiment, target cells expressing immunoproteasome can be transformed with the vector. The target cell may express immunoproteasome either constitutively, because of treatment with interferon (IFN), or through genetic manipulation, for example. CTL that recognize the mature epitope can be tested for their ability to recognize these target cells. In yet another embodiment, the embedded epitope can be prepared as a synthetic peptide. The synthetic peptide then can be subjected to digestion by an immunoproteasome preparation in vitro and the resultant fragments can be analyzed to determine the sites of cleavage. Such polypeptides, recombinant or synthetic, from which embedded epitopes can be successfully liberated, can also be incorporated into immunogenic compositions.

[0010] The invention disclosed herein relates to the identification of a polypeptide suitable for epitope liberation. One embodiment of the invention, relates to a method of identifying a polypeptide suitable for epitope liberation including, for example, the steps of identifying an epitope of interest; providing a substrate polypeptide sequence including the epitope, wherein the substrate polypeptide permits processing by a proteasome; contacting the substrate polypeptide with a composition including the proteasome, under conditions that support processing of the substrate polypeptide by the proteasome; and assaying for liberation of the epitope.

[0011] The epitope can be embedded in the substrate polypeptide, and in some aspects the substrate polypeptide can include more than one epitope, for example.
Also, the epitope can be a housekeeping epitope.

[0012] In one aspect, the substrate polypeptide can be a synthetic peptide.
Optionally, the substrate polypeptide can be included in a formulation promoting protein transfer.
Alternatively, the substrate polypeptide can be a fusion protein. The fusion protein can further include a protein domain possessing protein transfer activity. Further, the contacting step can include immunization with the substrate polypeptide.

[0013] In another aspect, the substrate polypeptide can be encoded by a polynucleotide. The contacting step can include immunization with a vector including the polynucleotide, for example. The immunization can be carried out in an HLA-transgenic mouse or any other suitable animal, for example. Alternatively, the contacting step can include transforming a cell with a vector including the polynucleotide. In some embodiments the transformed cell can be a target cell that is targeted by CTL for purposes of assaying for proper liberation of epitope.

[0014] The proteasome processing can take place intracellularly, either in vitro or in vivo. Further, the proteasome processing can take place in a cell-free system.

[0015] The assaying step can include a technique selected from the group including, but not limited to, mass spectrometry, N-terminal pool sequencing, HPLC, and the like. Also, the assaying step can include a T cell target recognition assay. The T cell target recognition assay can be selected from the group including, but not limited to, a cytolytic activity assay, a chromium release assay, a cytokine assay, an ELISPOT assay, tetramer analysis, and the like.

[0016] In still another aspect, the amino acid sequence of the substrate polypeptide including the epitope can be arbitrary. Also, the substrate polypeptide in which the epitope is embedded can be derived from an authentic sequence of a target-associated antigen. Further, the substrate polypeptide in which the epitope is embedded can be conformed to a preferred immune proteasome cleavage site flanking sequence.

[0017] In another aspect, the substrate polypeptide can include an array of additional epitopes. Members of the array can be arranged head-to-tail, for example. The array can include more than one housekeeping epitope. The more than one housekeeping epitope can include copies of the same epitope. The array can include a housekeeping and an immune epitope, or alternating housekeeping and immune epitopes, for example. Also, the array can include a housekeeping epitope positioned between two immune epitopes in an epitope battery. The array can include multiple epitope batteries, so that there are two immune epitopes between each housekeeping epitope in the interior of the array. Optionally, at least one of the epitopes can be truncated distally to its junction with an adjacent epitope. The truncated epitopes can be immune epitopes, for example. The truncated epitopes can have lengths selected from the group including, but not limited to, 9, 8, 7, 6, 5, 4 amino acids, and the like.

[0018) In still another aspect, the substrate polypeptide can include an array of epitopes and epitope clusters. Members of the array can be arranged head-to-tail, for example.

[0019] In yet another aspect, the proteasome can be an immune proteasome.
1 S [0020] Another embodiment of the disclosed invention relates to vectors including a housekeeping epitope expression cassette. The housekeeping epitope(s) can be derived from a target-associated antigen, and the housekeeping epitope can be liberatable, that is capable of liberation, from a translation product of the cassette by immunoproteasome processing.
[0021] In one aspect of the invention the expression cassette can encode an array of two or more epitopes or at least one epitope and at least one epitope cluster.
The members of the array can be arranged head-to-tail, for example. Also, the members of the array can be arranged head-to-tail separated by spacing sequences, for example. Further, the array can include a plurality of housekeeping epitopes. The plurality of housekeeping epitopes can include more than one copy of the same epitope or single copies of distinct epitopes, for example. The array can include at least one housekeeping epitope and at least one immune epitope. Also, the array can include alternating housekeeping and immune epitopes. Further, the array includes a housekeeping epitope sandwiched between two immune epitopes so that there are two immune epitopes between each housekeeping epitope in the interior of the array. The immune epitopes can be truncated distally to their junction with the adjacent housekeeping epitope.
[0022] In another aspect, the expression cassette further encodes an authentic protein sequence, or segment thereof, including at least one immune epitope.
Optionally, the segment can include at least one epitope cluster. The housekeeping epitope expression cassette and the authentic sequence including at least one immune epitope can be encoded in a single reading frame or transcribed as a single mRNA species, for example. Also, the housekeeping epitope expression cassette and the authentic sequence including at least one immune epitope may not be transcribed as a single mRNA species.

[0023) In yet another aspect, the vector can include a DNA molecule or an RNA
molecule. The vector can encode, for example, SEQ )D NO. 4, SEQ 117 NO. 17, SEQ )D NO. 20, SEQ ID NO. 26, SEQ Il7'NO. 27, SEQ ID NO. 29, SEQ ID NO. 33, and the like.
Also, the vector can include SEQ >I7 NO. 9, SEQ ID NO. 19, SEQ >D NO. 21, SEQ ID NO. 30, SEQ ID
NO. 34, and the like. Also, the vector can encode SEQ ID NO. 5 or SEQ I17 NO. 18, for example.
[0024] In still another aspect, the target-associated antigen can be an antigen derived from or associated with a tumor or an intracellular parasite, and the intracellular parasite can be, for example, a virus, a bacterium, a protozoan, or the like.
[0025] Another embodiment of the invention relates to vectors including a housekeeping epitope identified according to any of the methods disclosed herein, claimed or otherwise. For example, embodiments can relate to vector encoding a substrate polypeptide that includes a housekeeping epitope by any of the methods described herein.
[0026) In one aspect, the housekeeping epitope can be liberated from the cassette translation product by immune proteasome processing [0027] Another embodiment of the disclosed invention relates to methods of activating a T cell. The methods can include, for example, the steps of contacting a vector including a housekeeping epitope expression cassette with an APC. The housekeeping epitope can be derived from a target-associated antigen, for example, and the housekeeping epitope can be liberatable from a translation product of the cassette by immunoproteasome processing. The methods can further include contacting the APC with a T cell. The contacting of the vector with the APC can occur in vitro or in vivo.
[0028] Another embodiment of the disclosed invention relates to a substrate polypeptide including a housekeeping epitope wherein the housekeeping epitope can be liberated by immunoproteasome processing in a pAPC.
[0029] Another embodiment of the disclosed invention relates to a method of activating a T cell comprising contacting a substrate polypeptide including a housekeeping epitope with an APC wherein the housekeeping epitope can be liberated by immunoproteasome processing and contacting the APC with a T cell.
Brief Description of the Drawings [0030] Figure 1. An illustrative drawing depicting pMA2M.
[0031] Figure 2. Assay results showing the % of specific lysis of ELAGIGII,TV
pulsed and unpulsed T2 target cells by mock immunized CTL.
[0032) Figure 3. Assay results showing the % of specific lysis of ELAGIGILTV
pulsed and unpulsed T2 target cells by pVAXM3 immunized CTL.
[0033] Figure 4. Assay results showing the % of specific lysis of ELAGIGIL,TV
pulsed and unpulsed T2 target cells by pVAXM2 immunized CTL.

[0034] Figure S. Assay results showing the % of specific lysis of ELAGIGILTV
pulsed and unpulsed T2 target cells by pVAXMl immunized CTL.
[0035] Figure 6. Illustrates a sequence of SEQ m NO. 22 from which the NY-ESO-1 is~abs epitope is liberated by immunoproteasomal processing.
[0036] Figure 7. Shows the differential processing by immunoproteasome and housekeeping proteasome of the SLLMWITQC epitope (SEQ ID NO. 12) in its native context where the cleavage following the C is more efficiently produced by housekeeping than immunoproteasome.
[0037] Figure 8. 8A: Shows the results of the human immunoproteasome digest of SEQ 117 NO. 31. 8B: Shows the comparative results of mouse versus human immunoproteasome digestion of SEQ ID NO. 31.
[0038] Figure 9. Shows the differential processing of SSX-23,_68 by housekeeping and immunoproteasome.
Detailed Description of the Preferred Embodiment Definitions [0039] Unless otherwise clear from the context of the use of a term herein, the following listed terms shall generally have the indicated meanings for purposes of this description.
[0040] PROFESSIONAL ANTIGEN-PRESENTING CELL (pAPC) - a cell that possesses T cell costimulatory molecules and is able to induce a T cell response. Well characterized pAPCs include dendritic cells, B cells, and macrophages.
[0041] PERIPHERAL CELL - a cell that is not a pAPC.
[0042] HOUSEKEEPING PROTEASOME - a proteasome normally active in peripheral cells, and generally not present or not strongly active in pAPCs.
[0043] IMMUNOPROTEASOME - a proteasome normally active in pAPCs; the immunoproteasome is also active in some peripheral cells in infected tissues or following exposure to interferon.
[0044] EPITOPE - a molecule or substance capable of stimulating an immune response. In preferred embodiments, epitopes according to this definition include but are not necessarily limited to a polypeptide and a nucleic acid encoding a polypeptide, wherein the polypeptide is capable of stimulating an immune response. In other preferred embodiments, epitopes according to this definition include but are not necessarily limited to peptides presented on the surface of cells, the peptides being non-covalently bound to the binding cleft of class I MHC, such that they can interact with T cell receptors (TCR). Epitopes presented by class I MHC may be in immature or mature form. "Mature" refers to an MHC epitope in distinction to any precursor ("immature") that may include or consist essentially of a housekeeping epitope, but also includes other sequences in a primary translation product that are removed by processing, including without limitation, alone or in any combination, proteasomal digestion, N-terminal trimming, or the action of exogenous enzymatic activities. Thus, a mature epitope may be provided embedded in a somewhat longer polypeptide, the immunological potential of which is due, at least in part, to the embedded epitope; or in its ultimate form that can bind in the MHC binding cleft to be recognized by TCR, respectively.
[0045] MHC EPITOPE - a polypeptide having a known or predicted binding affinity for a mammalian class I or class II major histocompatibility complex (MHC) molecule.
[0046] HOUSEKEEPING EPITOPE - In a preferred embodiment, a housekeeping epitope is defined as a polypeptide fragment that is an MHC epitope, and that is displayed on a cell in which housekeeping proteasomes are predominantly active. In another preferred embodiment, a housekeeping epitope is defined as a polypeptide containing a housekeeping epitope according to the foregoing definition, that is flanked by one to several additional amino acids. In another preferred embodiment, a housekeeping epitope is defined as a nucleic acid that encodes a housekeeping epitope according to the foregoing definitions. Exemplary housekeeping epitopes are provide in U.S. Application No. 10/117,937, filed on April 4, 2002; and U.S. Provisional Application Nos. 60/282,211, filed on April 6, 2001; 60/337,017, filed on November 7, 2001;
60/363210 filed 3/7/02; and 60/409,123, filed on September 5, 2002; all of which are entitled EPITOPE SEQUENCES.
[0047) IMMUNE EPITOPE - In a preferred embodiment, an immune epitope is defined as a polypeptide fragment that is an MHC epitope, and that is displayed on a cell in which immunoproteasomes are predominantly active. In another preferred embodiment, an immune epitope is defined as a polypeptide containing an immune epitope according to the foregoing definition, that is flanked by one to several additional amino acids. In another preferred embodiment, an immune epitope is defined as a polypeptide including an epitope cluster sequence, having at least two polypeptide sequences having a known or predicted affinity for a class I MHC.
In yet another preferred embodiment, an immune epitope is defined as a nucleic acid that encodes an immune epitope according to any of the foregoing definitions.
[0048] TARGET CELL - a cell to be targeted by the vaccines and methods of the invention. Examples of target cells according to this definition include but are not necessarily limited to: a neoplastic cell and a cell harboring an intracellular parasite, such as, for example, a virus, a bacterium, or a protozoan. Target cells can also include cells that are targeted by CTL as a part of assays to determine or confirm proper epitope liberation and processing by a cell expressing immunoproteasome, to determine T cell specificity or immunogenicity for a desired epitope.. Such cells may be transfored to express the substrate or liberation sequence, or the cells can simply be pulsed with peptidelepitope.

[0049] TARGET-ASSOCIATED ANTIGEN (TAA) - a protein or polypeptide present in a target cell.
[0050] TUMOR-ASSOCIATED ANTIGENS (TuAA) - a TAA, wherein the target cell is a neoplastic cell.
[0051) HLA EPITOPE - a polypeptide having a known or predicted binding affinity for a human class I or class II HLA complex molecule.
[0052] ANTIBODY - a natural immunoglobulin (Ig), poly- or monoclonal, or any molecule composed in whole or in part of an Ig binding domain, whether derived biochemically or by use of recombinant DNA. Examples include inter alia, F(ab), single chain Fv, and Ig variable region-phage coat protein fusions.
[0053] ENCODE - an open-ended term such that a nucleic acid encoding a particular amino acid sequence can consist of codons specifying that (poly)peptide, but can also comprise additional sequences either translatable, or for the control of transcription, translation, or replication, or to facilitate manipulation of some host nucleic acid construct.
[0054] SUBSTANTIAL SIMILARITY - this term is used to refer to sequences that differ from a reference sequence in an inconsequential way as judged by examination of the sequence. Nucleic acid sequences encoding the same amino acid sequence are substantially similar despite differences in degenerate positions or modest differences in length or composition of any non-coding regions. Amino acid sequences differing only by conservative substitution or minor length variations are substantially similar. Additionally, amino acid sequences comprising housekeeping epitopes that differ in the number of N-terminal flanking residues, or immune epitopes and epitope clusters that differ in the number of flanking residues at either terminus, are substantially similar. Nucleic acids that encode substantially similar amino acid sequences are themselves also substantially similar.
[0055] FUNCTIONAL SIMILARITY - this term is used to refer to sequences that differ from a reference sequence in an inconsequential way as judged by examination of a biological or biochemical property, although the sequences may not be substantially similar. For example, two nucleic acids can be useful as hybridization probes for the same sequence but encode differing amino acid sequences. Two peptides that induce cross-reactive CTL
responses are functionally similar even if they differ by non-conservative amino acid substitutions (and thus do not meet the substantial similarity definition). Pairs of antibodies, or TCRs, that recognize the same epitope can be functionally similar to each other despite whatever structural differences exist.
In testing for functional similarity of immunogenicity one would generally immunize with the "altered" antigen and test the ability of the elicited response (Ab, CTL, cytokine production, etc.) 3 5 to recognize the target antigen. Accordingly, two sequences may be designed to differ in certain _g_ respects while retaining the same function. Such designed sequence variants are among the embodiments of the present invention.
[0056] EXPRESSION CASSETTE - a polynucleotide sequence encoding a polypeptide, operably linked to a promoter and other transcription and translation control elements, including but not limited to enhancers, termination codons, internal ribosome entry sites, and polyadenylation sites. The cassette can also include sequences that facilitate moving it from one host molecule to another.
[0057) EMBEDDED EPITOPE - an epitope contained within a longer polypeptide, also can include an epitope in which either the N- terminus or the C-terminus is embedded such that the epitope is not in an interior position.
[0058] MATURE EPITOPE - a peptide with no additional sequence beyond that present when the epitope is bound in the MHC peptide-binding cleft.
[0059] EPITOPE CLUSTER - a polypeptide, or a nucleic acid sequence encoding it, that is a segment of a native protein sequence comprising two or more known or predicted epitopes with binding affinity for a shared MHC restriction element, wherein the density of epitopes within the cluster is greater than the density of all known or predicted epitopes with binding affinity for the shared MHC restriction element within the complete protein sequence, and as disclosed in U.S.
Patent Application No. 09/561,571 entitled EPITOPE CLUSTERS.
[0060] Substrate or liberation sequence- a designed or engineered sequence comprising or encoding a housekeeping epitope (according to the first of the definitions offered above) embedded in a larger sequence that provides a context allowing the housekeeping epitope to be liberated by immunoproteasomal processing, directly or in combination with N-terminal trimming or other processes.
[0061) Degradation of cytosolic proteins takes place via the ubiquitin-dependent multi-catalytic multi-subunit protease system known as the proteasome. The proteasome degrades cytosolic proteins generating fragments that can then be translocated from the cytosol into the endoplasmic reticulum (ER) for loading onto class I MHC. Such protein fragments shall be referred to as class I peptides. The peptide loaded MHC are subsequently transported to the cell surface where they can be detected by CTL.
[0062] The mufti-catalytic activity of the proteasome is the result of its mufti-subunit structure. Subunits are expressed from different genes and assembled post-translationally into the proteasome complex. A key feature of the proteasome is its bimodal activity, which enables it to exert its protease, or cleavage function, with two discrete kinds of cleavage patterns. This bimodal action of the proteasome is extremely fundamental to understanding how CTL are targeted to recognize peripheral cells in the body and how this targeting requires synchronization between the immune system and the targeted cells.

[0063] The housekeeping proteasome is constitutively active in all peripheral cells and tissues of the body. The first mode of operation for the housekeeping proteasome is to degrade cellular protein, recycling it into amino acids. Proteasome function is therefore a necessary activity for cell life. As a corollary to its housekeeping protease activity, however, class I peptides generated by the housekeeping proteasome are presented on all of the peripheral cells of the body.
[0064] The proteasome's second mode of function is highly exclusive and occurs specifically in pAPCs or as a consequence of a cellular response to interferons (IFNs). In its second mode of activity the proteasome incorporates unique subunits, which replace the catalytic subunits of the constitutive housekeeping proteasome. This "modified"
proteasome has been called the immunoproteasome, owing to its expression in pAPC and as a consequence of induction by IFN
in body cells.
[0065] APC define the repertoire of CTL that recirculate through the body and are potentially active as killer cells. CTL are activated by interacting with class I peptide presented on the surface of a pAPC. Activated CTL are induced to proliferate and caused to recirculate through the body in search of diseased cells. This is why the CTL response in the body is defined specifically by the class I peptides produced by the pAPC. It is important to remember that pAPCs express the immunoproteasome, and that as a consequence of the bimodal activity of the proteasome, the cleavage pattern of proteins (and the resultant class I
peptides produced) are different from those in peripheral body cells which express housekeeping proteasome. The differential proteasome activity in pAPC and peripheral body cells, therefore, is important to consider during natural infection and with therapeutic CTL vaccination strategies.
[0066] All cells of the body are capable of producing IFN in the event that they are infected by a pathogen such as a virus. IFN production in turn results in the expression of the immunoproteasome in the infected cell. Viral antigens are thereby processed by the immunoproteasome of the infected cell and the consequent peptides are displayed with class I
MHC on the cell surface. At the same time, pAPC are sequestering virus antigens and are processing class I peptides with their immunoproteasome activity, which is normal for the pAPC
cell type. The CTL response in the body is being stimulated specifically by the class I peptides produced by the pAPC. Fortunately, the infected cell is also producing class I
peptides from the immunoproteasome, rather than the normal housekeeping proteasome. Thus, virus-related class I
peptides are being produced that enable detection by the ensuing CTL response.
The CTL immune response is induced by pAPC, which normally produce different class I peptides compared to peripheral body cells, owing to different proteasome activity. Therefore, during infection there is epitope synchronization between the infected cell and the immune system.
[0067] This is not the case with tumors and chronic viruses, which block the interferon system. For tumors there is no infection in the tumor cell to induce the immunoproteasome expression, and chronic virus infection either directly or indirectly blocks immunoproteasome expression. In both cases the diseased cell maintains its display of class I
peptides derived from housekeeping proteasome activity and avoids effective surveillance by CTL.
[0068) In the case of therapeutic vaccination to eradicate tumors or chronic infections, the bimodal function of the proteasome and its differential activity in APC
and peripheral cells of the body is significant. Upon vaccination with protein antigen, and before a CTL response can occur, the antigen must be acquired and processed into peptides that are subsequently presented on class I MHC on the pAPC surface. The activated CTL recirculate in search of cells with similar class I peptide on the surface. Cells with this peptide will be subjected to destruction by the cytolytic activity of the CTL. If the targeted diseased cell does not express the immunoproteasome, which is present in the pAPC, then the epitopes are not synchronized and CTL
fail to find the desired peptide target on the surface of the diseased cell.
[0069] Preferably, therapeutic vaccine design takes into account the class I
peptide that is actually present on the target tissue. That is, effective antigens used to stimulate CTL to 1 S attack diseased tissue are those that are naturally processed and presented on the surface of the diseased tissue. For tumors and chronic infection this generally means that the CTL epitopes are those that have been processed by the housekeeping proteasome. Tn order to generate an effective therapeutic vaccine, CTL epitopes are identified based on the knowledge that such epitopes are, in fact, produced by the housekeeping proteasome system. Once identified, these epitopes, embodied as peptides, can be used to successfully immunize or induce therapeutic CTL
responses against housekeeping proteasome expressing target cells in the host.
[0070] However, in the case of DNA vaccines, there can be an additional consideration. The immunization with DNA requires that APCs take up the DNA
and express the encoded proteins or peptides. It is possible to encode a discrete class I
peptide on the DNA. By immunizing with this construct, APCs can be caused to express a housekeeping epitope, which is then displayed on class I MHC on the surface of the cell for stimulating an appropriate CTL
response. Constructs for generation of proper termini of housekeeping epitopes have been described in U.S. Patent application No. 09/561,572 entitled EXPRESSION
VECTORS
ENCODING EPITOPES OF TARGET-ASSOCIATED ANTIGENS, filed on April 28, 2000.
[0071) Embodiments of the invention provide expression cassettes that encode one or more embedded housekeeping epitopes, and methods far designing and testing such expression cassettes. The expression cassettes and constructs can encode epitopes, including housekeeping epitopes, derived from antigens that are associated with targets. Housekeeping epitopes can be liberated from the translation products) of the cassettes. For example, in some embodiments of 3 5 the invention, the housekeeping epitope(s) can be flanked by arbitrary sequences or by sequences incorporating residues known to be favored in immunoproteasome cleavage sites.
In further embodiments of the invention multiple epitopes can be arrayed head-to-tail. In some embodiments, these arrays can be made up entirely of housekeeping epitopes. Likewise, the arrays can include alternating housekeeping and immune epitopes. Alternatively, the arrays can include housekeeping epitopes flanked by immune epitopes, whether complete or distally truncated.
In some preferred embodiments, each housekeeping epitope can be flanked on either side by an immune epitope, such that an array of such arrangements has two immune epitopes between each housekeeping epitope.
Further, the arrays can be of any other similar arrangement. There is no restriction on placing a housekeeping epitope at the terminal positions of the array. The vectors can additionally contain authentic protein coding sequences or segments thereof containing epitope clusters as a source of immune epitopes.
[0072] Several disclosures make reference to polyepitopes or string-of bead arrays.
See, for example, W00119408A1, March 22, 2001; W09955730A2, November 4, 1999;
W00040261A2, July 13, 2000; W09603144A1, February 8, 1996; EP1181314A1, February 27, 2002; W00123577A3, April 5; US6074817, June 13, 2000; US5965381, October 12, 1999;
W09741440A1, November 6, 1997; US6130066, October 10, 2000; US6004777, December 21, 1999; US5990091, November 23, 1999; W09840501A1, September 17, 1998;
W09840500A1, September 17, 1998; W00118035A2, March 15, 2001; W002068654A2, September 6, 2002;
W00189281A2, November 29, 2001; W00158478A, August 16, 2001; EP1118860A1, July 25, 2001; W00111040A1, February 15, 2001; W00073438A1, December 7, 2000;
W00071158A1, November 30, 2000; W00066727A1, November 9, 2000; W00052451A1, September 8, 2000;
W00052157A1, September 8, 2000; W00029008A2, May 25, 2000; W00006723A1, February 10, 2000. Additional disclosures, include Palmowski MJ, et al - J Immunol 2002;168(9):4391-8; Fang ZY, et al - Virology 2001;291(2):272-84; Firat H, et al - J Gene Med 2002;4(1):38-45; Smith SG, et al - Clin Cancer Res 2001;7(12):4253-61; Vonderheide RH, et al - Clin Cancer Res 2001;
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[0073] One important feature that the disclosures in the preceding paragraph all share is their lack of appreciation for the desirability of regenerating housekeeping epitopes when the construct is expressed in a pAPC. This understanding was not apparent until the present invention.
Embodiments of the invention include sequences, that when processed by an immune proteasome, liberate or generate a housekeeping epitope. Embodiments of the invention also can liberate or generate such epitopes in immunogenically effective amounts. Accordingly, while the preceding references contain disclosures relating to polyepitope arrays, none is enabling of the technology necessary to provide or select a polyepitope capable of liberating a housekeeping epitope by action of an immunoproteasome in a pAPC. In contrast, embodiments of the instant invention are based upon a recognition of the desirability of achieving this result. Accordingly, embodiments of the instant invention include any nucleic acid construct that encodes a polypeptide containing at least one housekeeping epitope provided in a context that promotes its generation via immunoproteasomal activity, whether the housekeeping epitope is embedded in a string-of beads array or some other arrangement. Some embodiments of the invention include uses of one or more of the nucleic acid constructs or their products that are specifically disclosed in any one or more of the above-listed references. Such uses include, for example, screening a polyepitope for proper liberation context of a housekeeping epitope and/or an immune epitope, designing an effective immunogen capable of causing presentation of a housekeeping epitope and/or an immune epitope on a pAPC, immunizing a patient, and the like. Alternative embodiments include use of only a subset of such nucleic acid constructs or a single such construct, while specifically excluding one or more other such constructs, for any of the purposes disclosed herein. Some preferred embodiments employ these and/or other nucleic acid sequences encoding polyepitope arrays alone or in combination. For example, some embodiments exclude use of polyepitope arrays from one or more of the above-mentioned references. Other embodiments may exclude any combination or all of the polyepitope arrays from the above-mentioned references collectively.
Some embodiments include viral and/or bacterial vectors encoding polyepitope arrays, while other embodiments specifically exclude such vectors. Such vectors can encode carrier proteins that may have some immunostimulatory effect. Some embodiments include such vectors with such immunostimulatory/immunopotentiating effects, as opposed to immunogenic effects, while in other embodiments such vectors may be included. Further, in some instances viral and bacterial vectors encode the desired epitope as a part of substantially complete proteins which are not associated with the target cell. Such vectors and products are included in some embodiments, while excluded from others. Some embodiments relate to repeated administration of vectors. In some of those embodiments, nonviral and nonbacterial vectors are included. Likewise, some embodiments include arrays that contain extra amino acids between epitopes, for example anywhere from 1-6 amino acids, or more, in some embodiments, while other embodiments specifically exclude such arrays.
[0074) Embodiments of the present invention also include methods, uses, therapies, and compositions directed to various types of targets. Such targets can include, for example, neoplastic cells such as those listed below, for example; and cells infected with any virus, bacterium, protozoan, fungus, or other agents, examples of which are listed below, in Tables 1-5, or which are disclosed in any of the references listed above. Alternative embodiments include the use of only a subset of such neoplastic cells and infected cells listed below, in Tables 1-5, or in any of the references disclosed herein, or a single one of the neoplastic cells or infected cells, while specifically excluding one or more other such neoplastic cells or infected cells, for any of the purposes disclosed herein. The following are examples of neoplastic cells that can be targeted:
human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia);
chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia);
and polycythemia vera, lymphoma (Hodgkin's disease and non Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, hepatocellular cancer, brain cancer, stomach cancer, liver cancer, and the like. Examples of infectious agents that infect the target cells can include the following: adenovirus, cytomegalovirus, Epstein-Barr virus, herpes simplex virus l, herpes simplex virus 2, human herpesvirus 6, varicella-zoster virus, hepatitis B virus, hepatitis D virus, papilloma virus, parvovirus B 19, polyomavirus BK, polyomavirus JC, hepatitis C virus, measles virus, rubella virus, human immunodeficiency virus (HIV), human T cell leukemia virus I, human T cell leukemia virus II, Chlamydia, Listeria, Salmonella, Legionella, Brucella, Coxiella, Rickettsia, Mycobacterium, Leishmania, Trypanasoma, Toxoplasma, Plasmodium, and the like.
Exemplary infectious agents and neoplastic cells are also included in Tables 1-5 below.

[0075] Furthermore the targets can include neoplastic cells described in or cells infected by agents that are described in any of the following references:
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[0076] Additional embodiments of the invention include methods, uses, therapies, and compositions relating to a particular antigen, whether the antigen is derived from, for example, a target cell or an infective agent, such as those mentioned above. Some preferred embodiments employ the antigens listed herein, in Tables 1-5, or in the list below, alone, as subsets, or in any combination. For example, some embodiments exclude use of one or more of those antigens.
Other embodiments may exclude any combination or all of those antigens.
Several examples of such antigens include MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGS, GAGE-1, GAGE-2, CEA, RAGE, NY-ESO, SCP-1, Hom/Mel-40, PRAMS, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Ban virus antigens, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CAM 17.1, NuMa, K-ras, (3-Catenin, CDK4, Mum-1, p16, as well as any of those set forth in the above mentioned references.
Other antigens are included in Tables 1-4 below.
[0077] Further embodiments include methods, uses, compositions, and therapies relating to epitopes, including, for example those epitopes listed in Tables 1-5. These epitopes can be useful to flank housekeeping epitopes in screening vectors, for example.
Some embodiments include one or more epitopes from Tables 1-5, while other embodiments specifically exclude one or more of such epitopes or combinations thereof.
Table 1 Virus Protein AA T cell epitope MHC molecule Position MHC ligand Anti en Adenovirus E3 9Kd 30-38 LIVIGILIL HLA-A*0201 SE . m N0.:44 Adenovirus EIA 234-243 SGPSNTPPEI H2-Db SE . )D N0.:45 Adenovirus E1B 192-200 VNIRNCCYI H2-Db (SE . ID N0.:46 Adenovirus EIA 234-243 SGPSNIPPEI T>I H2-Db SE . ID N0.:47 CSFV NS 2276-2284ENALLVALF SLA,haplotype of rotein d/d SE . ID N0.:48 Den a virusNS3 500-508 TPEGIIPTL HLA-B*3501 SE . ID N0.:49 EBV LMP-2 426-434 CLGGLLTMV HLA-A*0201 SE . ID N0.:50 EBV EBNA-1 480-484 NIAEGLRAL HLA-A*0201 SE .1D N0.:51 EBV EBNA-1 519-527 NLRRGTALA HLA-A*0201 SE . >D N0.:52 EBV EBNA-1 525-533 ALAIP CRL HLA-A*0201 SE .117 N0.:53 EBV EBNA-1 575-582 VLKDAIKDL HLA-A*0201 (SEQ. ID N0.:54) EBV EBNA-1 562-570 FMVFL THI HLA-A*0201 SE . m N0.:55 EBV EBNA-2 15-23 HLIVDTDSL HLA-A*0201 SE . B7 N0.:56 EBV EBNA-2 22-30 SLGNPSLSV HLA-A*0201 SE . ID N0.:57 EBV EBNA-2 126-134 PLASAMRML HLA-A*0201 SE . ID N0.:58) EBV EBNA-2 132-140 RMLWMANYI HLA-A*0201 SE . m N0.:59 EBV EBNA-2 133-141 MLWMANYN HL.A-A*0201 SE . ID N0.:60 EBV EBNA-2 151-159 ILPQGPQTA HLA-A*0201 SE . >D N0.:61 EBV EBNA-2 171-179 PLRPTAPTI HLA-A*0201 SE . ID N0.:62 EBV EBNA-2 205-213 PLPPATLTV HLA-A*0201 SE .1D N0.:63 EBV EBNA-2 246-254 RMHLPVLHV HLA-A*0201 SE . >D N0.:64 EBV EBNA-2 287-295 PMPLPPS L HLA-A*0201 (SEQ. ID N0.:65) EBV EBNA-2 294-302 LPPPAAPA HLA-A*0201 SE . ID N0.:66 EBV EBNA-2 381-389 SMPELSPVL HLA-A*0201 SE . ID N0.:67 EBV EBNA-2 453-461 DLDESWDYI HLA-A*0201 SE . ID N0.:68 EBV BZLFI 43-51 PLPCVLWPV HLA-A*0201 SE . ID N0.:69) EBV BZLFI 167-175 SLEECDSEL HLA-A*0201 SE . ID N0.:70 EBV BZLFl 176-t EIKRYKNRV HLA-A*OZOI

(SE . ID N0.:71) EBV BZLF1 195-203 QLL HYREV HLA-A*0201 (SE . ID N0.:72) EBV BZLFI 196-204 LL HYREVA HLA-A*0201 (SEQ. ID N0.:73 EBV BZLFI 217-225 LLK MCPSL HLA-A*0201 SE . 117 N0.:74 EBV BZLFI 229-237 SIIPRTPDV HLA-A*0201 SE . 117 N0.:75) EBV EBNA-6 284-293 LLDFVRFMGV HLA-A*0201 (SE . ID N0.:76 EBV EBNA-3 464-472 SVRDRLARL HLA-A*0203 SE . ID N0.:77 EBV EBNA-4 416-424 IVTDFSVIK HLA-A*1101 (SEQ. ID N0.:78 EBV EBNA-4 399-408 AVFDRKSDAK HLA-A*0201 (SE . B7 N0.:79 SE . ID N0.:80) SE . ID N0.:81) EBV EBNA-3 379-387 RPP1FIRRI. HLA-B7 SE . ID N0.:82 SEQ. ID N0.:83 SE . 117 N0.:84 SE . ID N0.:85 SE . ID N0.:86 EBV EBNA-1 72-80 R.P KRPSCI HLA-B7 SE . 117 N0.:87 SE . ID N0.:88) ( SEQ. ID N0.:89 SE . ID N0.:90 SE . ID N0.:91 SE . ID N0.:92 SE . ll~ N0.:93 SE . ID N0.:94 SE . ID N0.:9S

SE . ID N0.:96 (SE . ID N0.:97 SE . ID N0.:98 SE . 117 N0.:99 EBV LMP-2 236-244 RRRWRRLTV HLA-B*2704 SE . lD NO.:100 EBV EBNA-6 2S8-266 RRIYDLIEL HLA-B*2705 SE . ID NO.:101 EBV EBNA-3 4S8-466 YPLHE HGM HLA-B*3501 SEQ. )D NO.:102 EBV EBNA-3 4S8-466 YPLHE HGM HLA-B*3503 SEQ. ID NO.:103) SE . ID NO.:104 HCV env E 44-S1 ASRCWVAM HLA-B*3501 SE . 113 NO.: l OS

HCV core 27-3S G IVGGVYL HLA-B*40012 rotein SE . 117 N0.:106 HCV NSI 77-8S PPLTDFDQGW HLA-B*5301 SE . ID N0.:107 HCV core 18-27 LMGYIPLVGA H2-Dd rotein SEQ. ID NO.:108 HCV core 16-2S ADLMGYIPLV H2-Dd rotein SE . )D NO.:109 HCV NSS 409-424 MSYSWTGALVTPCAEE H2-Dd SE . ID NO.:110 HCV NS1 20S-213 KHPDATYSR Pa a-A06 SE . 117 NO.:111 HCV-1 NS3 400-409 KLVALGINAV HLA-A*0201 SEQ. ID NO.:112 HCV-1 NS3 440-448 GDFDSV~C Patr-B16 SE . )I7 N0.:113 HCV-1 env E 118-126 GNASRCWVA Patr-BI6 SE . ID NO.:114 HCV-1 NSI 1S9-167 TRPPLGNWF Patr-B13 SE . >D NO.:11 S

HCV-1 NS3 3S1-3S9 VPHPNIEEV Patr-B13 SE . ll~ NO.:116 HCV-1 ~~ NS3 438-446 YTGDFDSVI Patr-BO1 _ SE . ID N0.:117 HCV-1 NS4 328-335 SWAIKWEY Patr-All SEQ. ~ NO.:118 HCV-1 NSI 205-213 KHPDATYSR Patr-A04 SE . ID N0.:119 HCV-1 NS3 440-448 GDFDSVIDC Patr-A04 SEQ. ID N0.:120 SEQ. ID NO.:121 HIV a 24 267-275 IVGLNKNR HLA-A*3302 (SEQ. ID N0.:122) HIV a 24 262-270 EIYKRWIIL HLA-B8 (SEQ. ID NO.:123 HIV a 24 261-269 GEIYKRWII HLA-B8 SE . ID NO.:124 HIV a 17 93-101 EIKDTKEAL HLA-B8 SEQ. ID NO.:125 SEQ. ID N0.:126 HIV a 24 267-277 ILGLNKIVRMY HLA-B* 1501 (SEQ. ID N0.:127 SEQ. ILl N0.:128 HIV nef 115-125 YHTQGYFPQWQ HLA-B 17 SE . ID NO.:129 HIV nef 117-128 TQGYFPQWQNYT HLA-B17 SE . ID N0.:130 HIV I20 314-322 GRAFVT1GK HLA-B*2705 (SEQ. ID N0.:131 HIV a 24 263-271 KRWIII,GLN HLA-B*2702 SEQ. m NO.:132 HIV nef 72-82 VPLRPMTYK HLA-B*3501 SEQ. ID N0.:133 HIV nef 117-125 TQGYFP W HLA-B*3701 SE . ID NO.:134 HIV a 24 143-151 HQAISPRTI, HLA-Cw*0301 SEQ. ID NO.:135 HIV a 24 140-151 QMVH AISPRTL HLA-Cw*0301 SEQ, m N0.:136 HIV 120 431-440 MYAPPIGG I H2-Kd SE .117 N0.:137 HIV 160 318-327 RGPGRAFVTI H2-Dd SE . ll7 N0.:138 HIV 120 17-29 MPGRAFVTI H2-Ld SEQ. ID N0.:139 HIV-1 RT 476-484 LKEPVHGV HLA-A*0201 I

SE . ID N0.:140 HIV-1 nef 190-198 AFHHVAREL HLA-A*0201 SEQ. ID N0.:141 HIV-1 I60 120-128 KLTPLCVTL HLA-A*0201 SE . ID NO.:142 HIV-1 60 814-823 SLLNATDIAV HLA-A*0201 SE . ID NO.:143) HIV-1 RT 179-187 MY YMDDL HLA-A*0201 SE . 117 N0.:144 HIV-1 a 17 77-85 SLYNTVATL HLA-A*0201 SE . ID NO.:145) HIV-1 160 315-329 RGPGRAFVT1 HLA-A*0201 SE . ID NO.:146) SE .117 N0.:147 HIV-1 nef 73-82 VPLRPMTYK HLA-A3 SE . 117 NO.:148) SE . ID N0.:149) HIV-1 a 17 20-29 RLRPGGKKK HLA-A3 SE . ID NO.:150 SE . ID NO.:151 HIV-1 nef 74-82 VPLRPMTYK HLA-a* 11 SE . ID N0.:152 HIV-1 a 24 325-333 AIF SSMTK HLA-A*1101 SE . 117 NO.:153 HIV-1 nef 73-82 VPLRPMTYK HLA-A* 1101 SEQ. ID NO.:154 HIV-1 nef 83-94 AAVDLSHFLKEK HLA-A*1101 SE . ID NO.:155 HIV-1 a 24 349-359 AC GVGGPGGHK HLA-A*1101 SEQ. ID N0.:156 HIV-1 a 24 203-212 ETINEEAAEW HLA-A25 SE . ID N0.:157 HIV-1 nef 128-137 TPGPGVRYPL HLA-B7 SE . ID N0.:158 HIV-1 a 17 24-31 GGKKKYKL HLA-B8 SE . ID N0.:159 SEQ. ID NO.:160 HIV-1 a 24 298-306 DRFYKTLRA HLA-B 14 SE . II7 N0.:161 SE . ID NO.:162 HIV-1 a 24 265-24 KRWIII,GLNK HLA-B*2705 SEQ. ID NO.:163 HIV-1 nef 190-198 AFHHVAREL HLA-B*5201 SE . ll~ NO.:164 SE . 117 NO.:165 EBV EBNA-6 130-139 EENLLDFVRF HLA-B*4403 SE . ID NO.:166 _ I - ~ - - (SEQ. ID NO.:167) SE . m N0.:168 SE . >D N0.:169 HBV sA 348-357 GLSPTVWLSV HLA-A*0201 SE . ID NO.:170 HBV SA 335-343 WLSLLVPFV HLA-A*0201 SE . )D NO.:171 HBV cA 18-27 FLPSDFFPSV HLA-A*0201 SEQ. ID N0.:172) HBV cA 18-27 FLPSDFFPSV HLA-A*0202 SE . ID NO.:173 HBV cA 18-27 FLPSDFFPSV HLA-A*0205 SE . ID NO.:174 HBV cA 18-27 FLPSDFFPSV HLA-A*0206 SE . ID N0.:175) HBV of 575-583 FLLSLGIHL HLA-A*0201 SE . ID NO.:176 HBV of 816-824 SLYADSPSV HLA-A*0201 SE . ID NO.:177 HBV of 455-463 GLSRYVARL HLA-A*0201 SE . ID NO.:178 HBV env 338-347 LLVPFV WFV HLA-A*0201 SEQ. ID NO.:179) HBV of 642-650 ALMPLYACI HLA-A*0201 SEQ. ID NO.:180 HBV env 378-387 LLPIFFCLWV HLA-A*0201 SE . )D N0.:181 HBV of 538-546 YMDDVVLGA HLA-A*0201 SE . B7 NO.:182 HBV env 250-258 LLLCLIFLL HLA-A*0201 SEQ. ID N0.:183 HBV env 260-269 LLDYQGMLPV HLA-A*0201 SE . ID NO.:184 HBV env 370-379 SNSPFIPLL HLA-A*0201 SE . ID N0.:185 HBV env 183-191 FLLTRILTI HLA-A*0201 SE . ID NO.:186 HBV cA 88-96 YVNVNMGLK HLA-A* 1101 SEQ. ID N0.:187 HBV cA 141-151 STLPETTVVRR HLA-A*3101 SEQ. ID N0.:188 HBV eA 141-151 STLPETTVVRR HLA-A*6801 SE . ID N0.:189 HBV cA 18-27 FLPSDFFPSV HLA-A*6801 SE . ID NO.:190 HBV sA 28-39 IP SLDSWWTSL H2-Ld SEQ. )D NO.:191 HBV cA 93-100 MGLKFR L H2-Kb SE .117 NO.:l92 ~V ~ preS ~ 141-149~ STBXQSGXQ ~ HLA-A*0201 SE . ID N_O.:193 HCMV B 618-628 FIAGNSAYEYV HLA-A*0201 SE . ID N0.:194 SE . ID NO.:195 HCMV 65 397-411 DDVWTSGSDSDEELV HLA-b35 SE . ID NO.: I

HCMV 65 123-131 IPSINVHHY HLA-B*3501 SE . ID NO.:197) HCMV 65 495-504 NLVPMVATVO HLA-A*0201 SE . ID N0.:198 SE . ID NO.:199 HCV MP 17-25 DLMGYIPLV HLA-A*0201 SE . ID N0.:200 HCV MP 63-72 LLALLSCLTV HLA-A*0201 SEQ. ID N0.:201 HCV MP 105-112 ILHTPGCV HLA-A*0201 SE . ID N0.:202 HCV env E 66-75 LRRHIDLLV HLA-A*0201 SE . 117 N0.:203 HCV env E 88-96 DLCGSVFLV HLA-A*0201 SE . ID N0.:204 HCV env E 172-180 SMVGNWAKV HLA-A*0201 SEQ. ID N0.:205 HCV NSI 308-316 HLII NNDV HLA-A*0201 SE . ID N0.:206) HCV NSI 340-348 FLLLADARV HLA-A*0201 SEQ. ID N0.:207 HCV NS2 234-246 GLRDLAVAVEPVV HLA-A*0201 SE .11? N0.:208 HCV NSI 18-28 SLLAPGAKQNV HLA-A*0201 SE . D7 N0.:209 HCV NSI 19-28 LLAPGAK NV HLA-A*0201 SEQ. ID N0.:210 HCV NS4 192-201 LLFNILGGWV HLA-A*0201 SE . ID N0.:211 HCV NS3 579-587 YLVAY ATV HLA-A*0201 SE . ID N0.:212 HCV core 34-43 YLLPRRGPRL HLA-A*0201 rotein SE . ID N0.:213 HCV MP 63-72 LLALLSCLTI HLA-A*0201 SE . ID N0.:214) HCV NS4 174-182 SLMAFTAAV HLA-A*0201 SEQ. ID N0.:21 S

HCV NS3 67-75 CINGVCWTV HLA-A*0201 SE . ID N0.:216 HCV NS3 163-171 LLCPAGHAV HLA-A*0201 SE . ID N0.:217 HCV NS5 239-247 IL,DSFDPLV HLA-A*0201 SE .117 N0.:218 HCV NS4A 236-244 ILAGYGAGV HLA-A*0201 SE . ID N0.:219) HCV NSS 714-722 GL DCTMLV HLA-A*0201 (SE . ID N0.:220 HCV NS3 281-290 TGAPVTYSTY HLA-A*0201 SEQ. ID N0.:221 HCV NS4A 149-157 HMWNFISGI HLA-A*0201 SE . B7 N0.:222 HCV NSS 575-583 RVCEKMALY HLA-A*0201-A3 (SEQ. ID N0.:223) HCV NS1 238-246 TINYTIFK HLA-A*1101 SEQ. ID N0.:224) SEQ. ID N0.:225 HCV core 40-48 GPRLGVRAT HLA-B7 rotein SE .117 N0.:226 HIV-1 120 380-388 SFNCGGEFF HLA-Cw*0401 (SEQ. ID N0.:227 HIV-1 RT 206-214 TEMEKEGKI H2-Kk SE . ID N0.:228) HIV-1 17 18-26 KIRLRPGGK HLA-A*0301 SEQ. ID N0.:229 HIV-1 P17 20-29 RLRPGGKKKY HLA-A*0301 (SEQ. ID N0.:230 HIV- I RT 325-333 AIFQSSMTK HLA-A*0301 SE . ID N0.:231) SE . ID N0.:232 HIV-1 RT 508-517 IYQEPFKNLK HLA-All (SEQ. ID N0.:233 (SEQ. ID N0.:234) SEQ. ID N0.:235) HN-1 a 24 145-155 QAISPRTLNAW HLA-A25 SE . ID N0.:236 HN-1 a 24 167-175 EVIPMFSAL HLA-A26 SEQ. >I7 N0.:237 (SEQ. ID N0.:238 SEQ. ID N0.:239) SEQ. ID N0.:240 (SEQ. ID N0.:241 HIV-1 RT 71-79 ITLWQRPLV HLA-A*6802 SE . ID N0.:242 HIV-1 RT 85-93 DTVLEEMNL HLA-A*6802 SEQ.117 N0.:243 HIV-1 RT 71-79 ITLW RPLV HLA-A*7401 SE .1T7 N0.:244 HIV-1 a 24 148-156 SPRTLNAWV HLA-B7 SE . a? N0.:245 HIV-1 a 24 179-187 ATP DLNTM HLA-B7 SE .1D N0.:246 SE . >D N0.:247 HIV-1 41 ~ 843-8S1 IPRRIRQGL HLA-B7 SE . ~ N0.:248 SE . m N0.:249 HIV-1 nef 13-20 WPTVRERM HLA-B8 SE . ID N0.:2S0 HIV-1 nef 90-97 FLKEKGGL HLA-B8 SE . ID N0.:2S 1 ~-1 a 24 183-191 DLNTMLNTV HLA-B14 SE . B7 N0.:252 SE . ID N0.:253 SE .1D N0.:254 SE . ID N0.:255 HN-1 nef 73-82 VPLRPMTYK HLA-B27 SE . m N0.:2Sb SE . ID N0.:2S7 HIV-1 nef 105-114 RR DILDLWI HLA-B*2705 SE . ~ N0.:2S8) HIV-1 nef 134-141 RYPLTFGW HLA-B*2705 SEQ. ID N0.:2S9 SEQ. m N0.:260 SE . lZ7 N0.:261 ~-1 120 42-S2 VPVWKEATTTL HLA-B3S

SE . m N0.:262 SE . ID N0.:263 HIV-1 a 24 2S4-262 PPIPVGDIY HLA-B35 SE , m N0.:264 SE . ~ N0.:265) SE . ID N0.:266 HIV-1 a 2 45-253 NPVPVGNIY HLA-B3S

SE . ll~ N0.:267 ~-1 n ef 1 20-128 FPDWQNYT HLA-B37 Y

SE . ID N0.:268 HIV-1 a 24 1 93-201 H AAM ML HLA-B42 G

SE . m N0.:269 WO 2003/063770 PCT/US2002/036(19R

SE . ID N0.:270) SE . ID N0.:271 SE . ID N0.:272) _ HN-1 a 24 325-333 NANPDCKTI HLA-BS I

SE . ID N0.:273 HN-1 a 24 275-282 RMYSPTSI HLA-BS2 SEQ. ID N0.:274) HN-I 120 42-S1 VPVWKEATTT HLA-B*5501 SE . ID N0.:275) HN-I a 24 147-1SS ISPRTLNAW HLA-B57 (SE . ID N0.:276 HN-1 a 24 240-249 TSTL E IGW HLA-BS7 (SE .117 N0.:277) HIV-I a 24 162-172 KAFSPEVIPMF HLA-BS7 SE . ID N0.:278 HIV-1 a 24 311-319 ASQEVKNW HLA-BS7 SE . ID N0.:279 HIV-1 a 24 311-319 QAS DVKNW HLA-B57 SEQ. ID N0.:280 HN-1 nef 116-12S HT GYFPDW HLA-BS7 SEQ. ID N0.:281) HIV-1 nef 120-128 YFPDWQNYT HLA-BS7 (SE .117 N0.:282 HIV-1 a 24 240-249 TSTLQEQIGW HLA-BS8 SE . ID N0.:283) SE . ID N0.:284 SEQ. m N0.:285 (SEQ. m N0.:286 SE .117 N0.:287) HN-1 nef 117-127 TQGYFPDW NY HLA-B62 SE .117 N0.:288) HIV-1 nef 84-91 AVDLSHFL HLA-B62 SEQ. >I? N0.:289 HIV-1 a 24 168-175 VIPMFSAL H LA-Cw*0102 ( SEQ. m N0.:290 F

SE . ll7 N0.:291 HN-1 120 3?S-383 FNCGGEFF H LA-B1S
S

SE . m N0.:292 HIV-1 n ef 1 36-14S LTFGWCYKL H LA-A*0201 P

SE . ID N0.:293 HIV-1 n ef 1 80-189 LEWRFDSRL H LA-A*0201 V

SE . ID N0.:294 HN-1 n ef 6 8-77 PVTP VPLR H LA-B7 F

WO 2003/063770 PCT/US2002/036098. .____, I SEQ. ID N0.:295 HIV-1 nef 128-137 TPGPGVRYPL HLA-B7 ~SEQ. ID N0.:296) HIV-1 a 24 308-316 AS EVKNW HLA-Cw*0401 SE . ID N0.:297 HIV-lIIIB RT 273-282 VPLDEDFRKY HLA-B35 SE . ID N0.:298 HIV-l IIIB RT 25-33 NPDIVIY Y HLA-B35 SE . ID N0.:299 HIV-l IIIB 41 557-565 RAIEA AHL HLA-B51 SE . >D N0.:300) HIV-lIIIB RT 231-238 TAFTIPSI HLA-B51 SE . >I? NO.:301 HIV- I IIIB 24 215-223 VHPVHAGPIA HLA-B*5501 SE . ID N0.:302 HN-IIIIB 120 156-165 NCSFNISTSI HLA-Cw8 SE . ID N0.:303 HN- I IIIB 120 241-249 CTNVSTV C HLA-Cw8 SE . >I7 N0.:304 HIV-15F2 120 312-320 IGPGRAFHT H2-Dd SEQ. ID N0.:305 HIV-15F2 0l 25-33 NPDIVIY Y HLA-B*3501 SEQ. >D N0.:306 HIV-15F2 0l 432-441 EPIVGAETFY HLA-B*3501 SE . >D N0.:307 HIV-15F2 0l 432-440 EPNGAETF HLA-B*3501 SE . ID N0.:308 HIV-15F2 0l 6-14 SPAIFQSSM HLA-B*3501 SEQ. ID N0.:309 HIV-15F2 0l 59-68 VPLDKDFRKY HLA-B*3501 (SEQ. II? N0.:310 HIV-15F2 0l 6-14 IPLTEEAEL HLA-B*3501 SEQ. )D N0.:311 HIV-15F2 nef 69-79 RP VPLRPMTY HLA-B*3501 SEQ. m N0.:312 HIV-1 SF2 nef 66-74 FPVRP VPL HLA-B*3501 SE . m N0.:313 HIV-15F2 env 10-18 DPNPQEVVL HLA-B*3501 SEQ. ID N0.:314 HIV-1 SF2 env 7-15 RPIVSTQLL HLA-B*3501 SEQ. >D N0.:315 HIV-15F2 0l 6-14 IPLTEEAEL HLA-B51 SE . >D N0.:316 HN-15F2 env 10-18 DPNPQEVVL HLA-B51 SEQ. ID N0.:317 HIV-1 SF2 a 24 199-207 AMQMLKETI H2-Kd SEQ. >D N0.:318 HN-2 a 24 182-190 TPYDrNQML HLA-B*5301 SE .1D N0.:319 HN-2 a 260-269 RRWI LGL KV HLA-B*2703 SEQ.1D N0.:320 SE . ID N0.:321 SE . 117 N0.:322 HPV 6b E7 21-30 GLHCYE LV HLA-A*0201 SE . ID N0.:323 HPV 6b E7 47-55 PLK HF IV HLA-A*0201 SE . ID N0.:324 HPV 11 E7 4-12 RLVTLKDIV HLA-A*0201 SE . ID N0.:325 HPV 16 E7 86-94 TLGIVCPIC HLA-A*0201 SE .117 N0.:326 HPV 16 E7 85-93 GTLGIVCPI HLA-A*0201 SE . >D N0.:327 HPV 16 E7 12-20 MLDL PETT HLA-A*0201 SE . ID N0.:328 HPV16 E7 11-20 YMLDL PETT HLA-A*0201 SE . ID N0.:329 HPVl6 E6 15-22 RPRKLPQL HLA-B7 SE . ID N0.:330 HPV 16 E6 49-57 RAHYNIVTF HW-Db SE . ID N0.:331 HSV B 498-505 SSIEFARL H2-Kb SE . 117 N0.:332 HSV-1 C 480-488 GIGIGVLAA HLA-A*0201 SEQ. ID N0.:333 HSV-1 ICP27 448-456 DYATLGVGV H2-Kd SEQ. ID N0.:334) Virus Protein T cell epitope MHC molecule MHC ligand Anti en HSV-1 ICP27 322-332 LYRTFAGNPRA H2-Kd SEQ. ID N0.:335 HSV-1 UL39 822-829 TFDFGRL H2-Kb SE . ID N0.:336 HSV-2 C 446-454 GAGIGVAVL HLA-A*0201 SE . ID N0.:337) HLTV-1 TAX 11-19 LLFGYPVYV HLA-A*0201 SE . ID N0.:338 Influenza MP 58-66 GILGFVFTL HLA-A*0201 SEQ.117 N0.:339 Influenza MP 59-68 ILGFVFTLTV HLA-A*0201 SEQ. ID N0.:340) Influenza NP 265-273 ILRGSVAHK HLA-A3 SE . ID N0.:341 Influenza NP 91-99 KTGGPIYKR HLA-A*6801 SE .117 N0.:342 Influenza NP 380-388 ELRSRYWAI HLA-B8 SE . ID N0.:343) Influenza NP 381-388 LRSRYWAI HLA-B*2702 SE . D7 N0.:344 Influenza NP 339-347 EDLRVLSFI HLA-B*3701 SE .117 N0.:345 Influenza NSI 158-166 GEISPLPSL HLA-B44 SEQ. ID N0.:346 Influenza NP 338-346 FEDLRVLSF HLA-B44 SE . ID N0.:347 Influenza NSI 158-166 GEISPLPSL HLA-B*4402 SE . ID N0.:348 Influenza NP 338-346 FEDLRVLSF HLA-B*4402 SEQ.117 N0.:349 Influenza PBI 591-599 VSDGGPKLY . HLA-Al SE . ID N0.:350 Influenza NP 44-52 CTELKLSDY HLA-Al A

SE . ID N0.:351 Influenza NSI 122-130 AIMDKNIIL HLA-A*0201 SE . ID N0.:352 Influenza NSI 123-132 IMDKNIILKA HLA-A*0201 A

SE . ID N0.:353 Influenza NP 383-391 SRYWAIRTR HLA-B*2705 A

SEQ. ID N0.:354 Influenza NP 147-155 TY RTRALV H2-Kd A

SEQ. ID N0.:355 Influenza HA 210-219 TYVSVSTSTL H2-Kd A

SE . ID N0.:356 Influenza HA 518-526 IYSTVASSL H2-Kd A

SE . ID N0.:357 Influenza HA 259-266 FEANGNLI H2-Kk A

SEQ. ID N0.:358 Influenza HA 10-18 IEGGWTGMI H2-Kk A

SE . ID N0.:359) Influenza NP 50-57 SDYEGRLI H2-Kk A

(SEQ. ID N0.:360) Influenza NSI 152-160 EEGAIVGEI H2-Kk a SEQ. LD N0.:361 Influenza NP 336-374 ASNENMETM H2Db SE .117 N0.:362 Influenza NP 366-374 ASNENMDAM H2Db SEQ. ID N0.:363 Influenza NP 85-94 KLGEFYN MM HLA-A*0201 B

SEQ. ID N0.:364 Influenza NP 85-94 KAGEFYNQMM HLA-A*0201 B

SEQ. ID N0.:365 Influenza HA 204-212 LYQNVGTYV H2Kd JAP

SE . ID N0.:366 Influenza HA 210-219 TYVSVGTSTL H2-Kd JAP

(SEQ. ID N0.:367 Influenza HA 523-531 VYQILATYA H2-Kd JAP

SE . ID N0.:368 Influenza HA 529-537 IYATVAGSL H2-Kd JAP

SE . ID N0.:369 Influenza HA 210-219 TYVSVGTSTI L>I H2-Kd JAP

SE . ID N0.:370 Influenza HA . 255-262 FESTGNLI H2-Kk JAP

SE . ID N0.:371 JHMV cA 318-326 APTAGAFFF H2-Ld SE . ID N0.:372 LCMV NP 118-126 RP ASGVYM H2-Ld SE .117 N0.:373 LCMV NP 396-404 F P NGQFI H2-Db SEQ. ID N0.:374 LCMV GP 276-286 SGVENPGGYCL H2-Db (SEQ. ID N0.:375 LCMV GP 33-42 KAVYNFATCG H2-Db (SEQ. ID N0.:376 MCMV 89 168-176 YPHFMPTNL H2-Ld SE . >D N0.:377 MHV spike 510-518 CLSWNGPHL H2-Db rotein SE . ID N0.:378 MMTV env 36 474-482 SFAVATTAL H2-Kd SEQ. D7 N0.:379 MMTV a 27 425-433 SYETFISRL H2-Kd SE . ID N0.:380 MMTV env 73 544-551 ANYDFICV H2-Kb (SEQ. ID N0.:381 MuLV env 15E 574-581 KSPWFTTL H2-Kb SEQ. ID N0.:382 MuLV env gp70189-196 SSWDFITV H2-Kb SE . ID N0.:383 MuLV a 75K 75-83 CCLCLTVFL H2-Db (SE .117 N0.:384 MuLV env 70 423-431 SPSYVYHQF H2Ld SE . ID N0.:385 MV F rotein437-447 SRRYPDAVYLH HLA-B*2705 (SEQ. ID N0.:386 My F rotein438-446 RRYPDAVYL HLA-B*2705 Virus Protein AA T cell epitope MHC MHC molecule Position ligand Anti en (SE . ID N0.:387 My NP 281-289 YPALGLHEF H2-Ld (SE . ID N0.:388 My HA 343-351 DPV117RLYL H2-Ld SE . ID N0.:389 MV HA 544-552 SPGRSFSYF H2-Ld (SEQ. ID N0.:390 Poliovirus VP1 111-118 TYKDTV L H2-kd SE . ID N0.:391 Poliovirus VP1 208-217 FYDGFSKVPL H2-Kd SE . ID N0.:392 Pseudorabies6111 455-463 IAGIGILAI HLA-A*0201 virus SE . ID N0.:393 RabiesvirusNS 197-205 VEAEIAHQI H2-Kk SEQ. ID N0.:394 Rotavirus VP7 33-40 11YRFLL1 H2-Kb SE . ID N0.:395) Rotavirus VP6 376-384 VGPVFPPGM H2-Kb SE . ID N0.:396 Rotavirus VP3 585-593 YSGYIFRDL H2-Kb SE .117 N0.:397 RSV M2 82-90 SYIGSINNI H2-Kd SEQ. ID N0.:398 SN a 11 C 179-190 EGCTPYDTNQML Mamu-A*O1 SE . )17 N0.:399 SV NP 324-332 FAPGNYPAL H2-Db SEQ. ID N0.:400 SV NP 324-332 FAPCTNYPAL H2-Kb SE . ID N0.:401 SV40 T 404-411 VVYDFLKC H2-Kb SE . ID N0.:402) SV40 T 206-215 SAINNYA KL H2-Db SE . ID N0.:403 SV40 T 223-231 CKGVNKEYL H2-Db SE . ID N0.:404 SV40 T 489-497 GINNLDNL H2-Db SEQ. ID N0.:405 SV40 T 492-S00 NNLDNLRDY(L) H2-Db SEQ. ID N0.:406 SV40 T 560-568 SEFLLEKRI H2-Kk SEQ. ID N0.:407 VSV NP 52-59 RGYVY GL H2-Kb SEQ. ID N0.:408 Table 2 IEILA-A1 Position Anti en Source T cell a EADPTGHSY MAGE-1 161-169 ito es SEQ. ID N0.:409 VSDGGPNLY Influenza A PB 1591-599 SEQ. ID N0.:410 CTELKLSDY Influenza A NP 44-52 SEQ. ID N0.:411 SEQ. ff~ N0.:412 HLA-A201 MLLSVPLLLG Calreticulin si al se uence SEQ, )D N0.:413 SE . >D N0.:414 YMDGTMS V Tyrosinase 369-377 SE . ID N0.:415 SEQ. ID N0.:416 LLGFVFTLTV Influenza MP 59-68 SEQ. ID N0.:417 LLFGYPVYVV HTLV-1 tax 11-19 (SEQ. )D N0.:418 GLSPTVWLSV HBV sA 348-357 SEQ.117 N0.:419 WLSLLVPFV HBV sA 335-343 SE . ID N0.:420 FLPSDFFPSV HBV cA 18-27 SEQ. ID N0.:421) SEQ. ID N0.:422) (SEQ. )D N0.:423 KLGEFYNQMM Influenza BNP 85-94 SEQ. m N0.:424) SE . ID N0.:425) SE . ID N0.:426) (SE . )D N0.:427 MLLAVLYCL T rosinase 1-9 SE . II7 N0.:428) AAGIGILTV Melan A~IVIart-127-35 (SEQ. ID N0.:429 YLEPGPVTA Pmel 17/ 100 480-488 SEQ. ID N0.:430 ~ ILDGTATLRL Pmel 17/ gp 100 457-466 SE . ID N0.:431) LLDGTATLRL Pmel 100 457-466 SE . ID N0.:432 ITD VPFSV Pmel 100 209-217 SE . ID N0.:433 KTWGQYWQV Pmel 100 154-162 SE . ID N0.:434) TITDQVPFSV Pmel 100 208-217 SE . ID N0.:435) AFHIIVAREL HIV- I nef 190-198 SE . ID N0.:436) YLNKIQNSL P. falci arum CSP 334-342 SE . ID N0.:437 MMRKLAILSV P. falci arum CSP 1 -10 SE . ID N0.:438 KAGEFYN MM Influenza BNP 85-94 SE . ID N0.:439) SEQ. ID N0.:440) SE . ID N0.:441 SE . ID N0.:442) SE . ID N0.:443 SE . ID N0.:444) SEQ. ID N0.:445) SE . ID N0.:446) SE . ID N0.:447 SE . ID N0.:448 SE . ID N0.:449) SEQ. >D N0.:450 SE .117 N0.:451 SEQ.117 N0.:452) SE . ID N0.:453 SE . ID N0.:454 SE . ID N0.:455 SE . ID N0.:456 SE . ID N0.:457 SE . ID N0.:458 SEQ, ID N0.:459 SE . ID N0.:460 SE . ID N0.:461 SE . ID N0.:462 SE . ID N0.:463 SE . ID N0.:464 AIMDKNIIL Influenza A NS1 122-130 SE . ID N0.:465 IMDKNIILKA Influenza A NSl 123-132 SE . ID N0.:466 SE . ID N0.:467 SE . ID N0.:468 LRRHIDLLV HCV env E 66-75 SEQ, ID N0.:469 DLCGSVFLV HCV env E 88-96 SEQ. ID N0.:470 SMVGNWAKV HCV env E 172-180 SE . ID N0.:471 SE . ID N0.:472 SEQ. ID N0.:473 SEQ. ID N0.:474 SE .117 N0.:475 LLAPGAK NV HCV NSl 19-28 SE . ID N0.:476 SE . ID N0.:477 SE .117 N0.:478 SEQ. >D N0.:479 SE . 117 N0.:480 SE . >D N0.:481 (SE . ID N0.:482 SE . ID N0.:483 VLYRYGSFSV Pmel 100 476-485 SE . >D N0.:484 YIGEVLVSV Non-filament forming class I myosin family (HA-2)**

(SE . ID N0.:485 SE . ID N0.:486 LLVPFV WFW HBV env 338-347 SE . ID N0.:487 SE . ID N0.:488 (SE . ID N0.:489 (SEQ. )D N0.:490 YLLPRRGPRL HCV core rotein 34-43 SE . ID N0.:491 LLPIFFCLWV HBV env 378-387 SE . ID N0.:492 YMDDVVLGA HBV Pol 538-546 SE . ID N0.:493 SE . ID N0.:494 (SEQ. ID N0.:495 SE . ID N0.:496 SE . ID N0.:497 SE . )D N0.:498 VMNILL YVV Glutarnic acid decarbox lase 114-123 SE . >D N0.:499 ILTVILGVL Melan A/Mart- 32-40 SE . ID NO.:500 SE . >D NO.:501 SE . ID N0.:502 IL,DSFDPLV HCV NSS 239-247 SE . ID N0.:503 LLLCLIFLL HBV env 250-258 (SEQ. m N0.:504 LIDYQGMLPV HBV env 260-269 SEQ. ID N0.:505 SIVSPFIPLL HBV env 370-379 SE . ID N0.:506 FLLTRILTI HBV env 183-191 (SE . ID N0.:507 HLGNVKYLV P. faci arum TRAP 3-11 SE . ID N0.:508 GIAGGLALL P. faci arum TRAP 500-508 SE . ID N0.:509 SEQ.117 N0.:510 SE .117 N0.:511 (SEQ. ID N0.:512 SEQ. ID N0.:513) VLPDVFIRCV N-acetylglucosaminyltransferase V
Gnt-V intron SEQ. ID N0.:514 VLPDVFIRC N-acetylglucosaminyltransferase V
Gnt-V intron SEQ. ID N0.:515 AVGIGIAVV Human CD9 SEQ. ID N0.:516 LVVLGLLAV Human lutam ltransferase SE . ID N0.:517 ALGLGLLPV Human G rotein cou led rece for (SEQ. ID N0.:518) SEQ. ID N0.:519 SE .117 N0.:520 IAGIGILAI Pseudorabies GIN 455-463 SEQ.117 N0.:521 LIVIGILIL Adenovirus 3 E3 9kD 30-38 SEQ. ID N0.:522 LAGIGLIAA S. Lincolnensis ImrA

SE . ID N0.:523 VDGIGILTI Yeast sa-177-85 SEQ. ID N0.:524 GAGIGVLTA B. polymyxa, [3cndoxylanase (SEQ. ll7 N0.:525) AAGIGII I E. coli methionine s thane SEQ. ID N0.:526 AGIGILLA E. coli h othetical rotein SEQ. ID N0.:527 KARDPHSGHFV CDK4wl 22.32 SEQ. ID N0.:528 SEQ. ID N0.:529 SEQ. ID N0.:530 SLYNTVATL HIV- I a 17 77-85 SEQ. ID N0.:531 ELVSEFSRV HER-2, m>V substituted SE . ID N0.:532 SEQ. ID N0.:533 SEQ. ID N0.:534 SEQ. ID N0.:535 GLHCYEQLV HPV 6b E7 21-30 SEQ. ID N0.:536 PLKQHFQIV HPV 6b E7 47-55 SE . ID N0.:537 SE . >I7 N0.:538 AIMEKNIML Influenza Alaska NS 1 122-130 SEQ. ID N0.:539 YLKTI NSL P. falci arum c 36 CSP

SEQ. ID N0.:540 YLNKIQNSL P. falci arum c 39 CSP

SEQ. ID N0.:541 YMLDL PETT HPV 16 E7 11-20*

SE .117 N0.:542 LLMGTLGIV HPV16 E7 82-90**

SE . ID N0.:543 SEQ. ID N0.:544 SE . ID N0.:545 SEQ. ID N0.:546 TIH>DIIL,EC HPV 16 E6 29-37 SEQ. ID N0.:547 SE . ID N0.:548) VIL,GVLLLI Melan A/Mart-1 35-43 SE . ID N0.:549 ALMDKSLHV Melan A/Mart- 1 56-64 SE . ID N0.:550 GIL,TVILGV Melan A/Mart- 1 31-39 SE . ID NO.:551 T cell a ito MINAYLDKL P. Falci arum STARP 523-531 es SE . ID N0.:552) AAGIGILTV Melan A/Mart- 127-35 SE . ID N0.:553 FLPSDFFPSV HBV cA 18-27 SE . ID N0.:554 Motif unknownSVRDRLARL EBNA-3 464-472 T cell a ito SE . ID NO.:555 es T cell a ito AAGIGILTV Melan A/Mart-1 27-35 es SE . ID N0.:556 FAYDGKDYI Human MHC I-of 140-148 SE . )D N0.:557 T cell a ito AAGIGILTV Melan A/Mart-1 27-35 es SE . ID N0.:558 FLPSDFFPSV HBV cA 18-27 SE . ID N0.:559 Motif unknownAAGIGILTV Meland A/Mart-1 27-35 T cell a ito SE . ID N0.:560 es FLPSDFFPSV HBV cA 18-27 SEQ. ID N0.:561 AAGIGILTV Melan A/Mart-1 27-35 SE . ID N0.:562 ALLAVGATK Pmel17 100 17-25 SE . ID N0.:563 T cell a ito R L R D L L L I V HIV-1 41 768-778 es T R

SE . ID N0.:564 VPLRPMTYK HIV-1 nef 73-82 SEQ. ID N0.:565) SE . ID N0.:566) RLRPGGKKK HIV- 1 a 17 20-29 SE . ID N0.:567 ILRGSVAHK Influenza NP 265-273 SE . ID N0.:568 SEQ. ID N0.:569 SE .117 N0.:570 SE . ID N0.:571 SE . ID N0.:572 Motif unknownKIFSEVTLK Unknown; muta melanoma peptide ted I83L 1?5-183 T cell a SEQ. ID N0.:573 ito a YVNVNMGLK* HBV cA 88-96 (SE . ID N0.:574 T cell a IVTDFSVIK EBNA-4 416-424 ito es SEQ. 117 N0.:575) SEQ. ID N0.:576) (SE . ID N0.:577 AIF SSMTK HIV- I a 24 325-333 SE .117 N0.:578) QVPLRPMTYK HIV-1 nef 73-82 SE . ID N0.:579 SE . ID N0.:580) AAVDLSHFLKEK HIV-1 nef 83-94 (SE . ID N0.:581 K

SE . ID N0.:581 HLA-A24 S Y L D S G I H F* ~3-catenin, mutated (proto-onocogen) SE . 117 N0.:582 T cell a RYLKDQQLL HIV GP 41 583-591 ito es SE . ID N0.:583) AYGLDFYIL, P15 melanoma A 10- 18 SE . ID N0.:584) AFLPWHRLFL T osinase 206-215 SE . ID N0.:585) AFLPWHRLF Tyrosinase 206-214 SE . ID N0.:586 RYSIFFDY Ebna-3 246-253 SE . ID N0.:587 T cell a ETINEEAAEW HIV- 1 a 24 203-212 ito a SE . ID N0.:588 T cell a STLPETTVVRR HBV cA 141 -151 ito es SE . ID N0.:589 MSL R FLR ORF 3P- 75 294-321 b SE . ID N0.:590 LLPGGRPYR TRP t osinase rel. 197-205 SE . ID N0.:591 T cell a IVGLNKIVR HIV a 24 267-267-275 ito a SE . ID N0.:592 AAGIGILTV Melan A/Mart- 127 35 SE . ID N0.:593 Table 3 sets forth additional antigens useful in the invention that are available from the Ludwig Cancer Institute. The Table refers to patents in which the identified antigens can be found.
TRA refers to the tumor-related antigen and the LUD No. refers to the Ludwig Institute number.
Table 3 TRA LUD Patent Date Patent Peptide (Antigen) HLA
No. No. Issued MAGE-4 5293 5,405,94011 April EVDPASNTY HLA-AI

(SEQ. ID N0.:532) MAGE-41 5293 5,405,94011 April EVDPTSNTY HLA-A

(SEQ ID N0:533) MAGE-5 5293 5,405,94011 April EADPTSNTY HLA-A

(SEQ ID N0:534) MAGE-51 5293 5,405,9401 1 April EADPTSNTY HLA-A

(SEQ ID N0:534) MAGE-6 5294 5,405,940I 1 April EVDPIGHVY HLA-AI

(SEQ ID N0:535) 5299.25,487,97430 January MLLAVLYCLL HLA-A2 (SEQ ID N0:536) 5360 5,530,09625 June 1996MLLAVLYCL HLA-B44 (SEQ ID N0:537) Tyrosinase5360.15,519,11721 May 1996 SEIWRDIDFA HLA-B44 (SEQ ID N0:538) SEIWRDIDF

(SEQ ID N0:539) Tyrosinase5431 5,774,31628 April XEIWRDIDF HLA-B44 (SEQ ID N0:540) MAGE-2 5340 5,554,72410 SeptemberSTLVEVTLGEV HLA-A2 (SEQ ID N0:541) LVEVTLGEV

(SEQ ID N0:542) V IFSKASEYL

(SEQ ID N0:543) IIVLAIIAI

(SEQ ID N0:544) (Continued) KIWEELSMLEV

(SEQ ID N0:545) TRA LUD Patent Date Patent Peptide (Antigen) HLA
No. No. Issued LIETSYVKV

(SEQ ID N0:546) 5327 5,585,46117 December FLWGPRALV HLA-A2 (SEQ ID NO: 547) TLVEVTLGEV

(SEQ ID N0:548) ALV ETSYV KV

(SEQ ID N0:549) MAGE-3 5344 5,554,50610 SeptemberK(WEELSVL HLA-A2 (SEQ ID N0:550) MAGE-3 5393 5,405,94011 April EVDPIGHLY HLA-Al (SEQ ID N0:551) MAGE 5293 5,405,94011 April EXDXSY HLA-Al (SEQ. ID N0.:552) (but not EADPTGHSY) (SEQ. ID N0.:553) E (A/V) D X5 Y

(SEQ. ID N0.:554) E(A/V)DPX4Y

(SEQ. ID N0.:555) E (A/V) D P (1/A/T) (SEQ. ID N0.:556) E (A/V) D P (I/A/T) (G/S) X2 Y

(SEQ. ID N0.:557) E (A/V) D P (1/A/T) (G/S) (H/N) X Y

E (A/V) DP (I/A/T) (G/S) (H/N) (L/T/V) Y

(SEQ. I I) N0.:559) MACE-I 5361 5,558.99524 SeptemberELHSAYGEPRKLLTQD HLA-C

(SEQ ID N0:560) Clone EHSAYGEPRKLL

(SEQ ID N0:561 ) SAYGEPRKL

(SEQ ID N0:562) MAGE-1 5253.4TBA TBA EADPTGHSY HLA-A
I

(SEQ ID N0:563) TRA LUD Patent Date Patent Peptide (Antigen) HLA
No. No. Issued BAGS 5310.1TBA TBA MAARAVFLALSAQLLQARLMKEHLA-C

(SEQ ID N0:564) Clone MAARAVFLALSAQLLQ HLA-C

(SEQ ID N0:565) Clone AARAVFLAL HLA-C

(SEQ ID N0:566) Clone GAGE 5323.25,648,22615 July 1997YRPRPRRY HLA-CW6 (SEQ. ID N0.:567) Table 4 Source Protein AA MHC T cell epitope Ref.
PositionmoleculesMHC
li and Anti en syntheticsyntheticsyntheticHLA-A2 ALFAAAAAV Parker, et peptides peptidespeptides al., - "Scheme for ranking potential HLA-A2 binding peptides based onindependent binding of individual peptide side-chains,"
J.
Immunol.
152:163-175 " GIFGGVGGV "

" GLDKGGGV "

" GLFGGFGGV "

" GLFGGGAGV "

" GLFGGGEGV "

" GLFGGGFGV "

" GLFGGGGGL "

" GLFGGGGGV "

" GLFGGGVGV "

" GLFGGVGGV "

" GLFGGVGKV "

" GLFKGVGGV "

" GLGGGGFGV "

" GLLGGGVGV "

" GLYGGGGGV "

" GMFGGGGGV "

" GMFGGVGGV "

" GQFGGVGGV "

" GVFGGVGGV "

" KLFGGGGGV "

" KLFGGVGGV "

" AILGFVFTL "

" GAIGFVFTL "

" GALGFVFTL "

" GELGFVFTL "

" GIAGFVFTL "

" GIEGFVFTL "

" GILAFVFTL "

" GILGAVFTL "

" GILGEVFTL "

" GILFGAFTL "

" GILGFEFTL "

" GILGFKFTL "

" GILGFVATL "

" GILGFVETL "

" GILGFVFAL "

" GIL,GFVFEL "

" GILGFVFKL "

" GILGFVFTA "

" GILGFVFTL "

" GILGFVFVL "

" GILGFVKTL "

" GILGKVFTL "

" GILKFVFTL "

" GILPFVFTL "

" GIVGFVFTL "

" GKLGFVFTL "

" GLLGFVFTL "

" G LGFVFTL "

" KALGFVFTL "

" KILGFVFTL "

" KILGKVFTL "

" AILLGVFML "

" AlYKRWIIL "

" ALFFFDIDL "

" ATVELLSEL "

" CLFGYPVYV "

" FIFPNYTIV "

" IISLWDS L "

" ILASLFAAV "

" ILESLFAAV "

" KLGEFFN M "

" KLGEFYN M "

" LLFGYPVYV "

" LLWKGEGAV "

" LMFGYPVYV "

" LNFGYPVYV "

" LQFGYPVYV "

" NIVAHTFKV "

" NLPMVATV "

" QMLLAIARL "

" QMWQARLTV "

" RLLQTGIHV "

" RLVNGSLAL "

" SLYNTVATL "

" TLNAWVKVV "

" WLYRETCNL j " YLFKRMIDL "

" GAFGGVGGV "

" GAFGGVGGY "

" GEFGGVGGV "

" GGFGGVGGV "

" GIFGGGGGV "

" GIGGFGGGL "

" GIGGGGGGL "

" GLDGGGGGV "

" GLDGKGGGV "

" GLDKKGGGV "

" GLFGGGFGF "

" GLFGGGFGG "

" GLFGGGFGN "

" GLFGGGFGS "

" GLFGGGGGI "

" GLFGGGGGM "

" GLFGGGGGT "

" GLFGGGGGY "

" GLGFGGGGV "

" GLGGFGGGV "

" GLGGGFGGV "

" GLGGGGGFV "

" GLGGGGGGY "

" GLGGGVGGV "

" GLLGGGGGV "

" GLPGGGGGV "

" GNFGGVGGV "

" GSFGGVGGV "

" GTFGGVGGV "

" AGNSAYEYV "

" GLFPGQFAY "

" HILLGVFML "

" ILESLFRAV "

" KKKYKLKHI "

" MLASIDLKY "

" MLERELVRK "

" KLFGFVFTV "

" ILDKKVEKV "

" ILKEPVHGV "

" ALFAAAAAY "

" GIGFGGGGL "

" GKFGGVGGV "

" GLFGGGGGK "

" EILGFVFTL "

" GIKGFVFTL "

" GQLGFVFTK I "

" ILGFVFTLT "

" KII,GFVFTK "

" KKLGFVFTL "

" KLFEKVYNY "

" LRFGYPVYV "

Human HSP60 140-148 HLA-B27 IRRGVMLAV Rammensee et al. I 997 " " 369-377 " KRI EIIEQ "

" " 469-477 " KRTLKIPAM "

YersiniaHSP60 35-43 " GRNVVLDKS "

" " 117-125 " KRGIDKAVI "

" " 420-428 " IRAASAITA "

" HSP 60 284-292 HLA- RRKAMFEDI 169 B*2705 P.falci LSA-1 1850-1857HLA-B3501KPKDELDY 170 arum Influenza 379-387 HLA- LELRSRYWA 183 NP B *4402 Tum-P35B 4-13 HLA-Dd GPPHSNNFGY 230 RotavirusVP7 33-40 IIYRFLLI 262 OGDH 104-112 H2-Ld QLSPYPFDL 253 TRP-2 181-188 p287 VYDFFVWL 284 DEAD box 547-554 p287 SNFVFAGI 283 Vector p287 SVVEFSSL 260 "artefact"

Epiope p287 AHYLFRNL 278 mimic of tumor A

" THYLFRNL "

Epitope " LNIYNTL 279 mimic of H-3 miHA
"

" LIYEFNTL "

" IPYIYNTL "

" IIYII'IIRL "

" LIYIFNTL "

HBV cA 93-100 " MGLKFR L 280 Human autoantigen51-58 " 1MIKFRNRL 281 LA

Mouse UTY H2Db WMHIINMDLI 303 rotein Mouse 53 232-240 " KYMCNSSCM 302 MURINE MDM2 441-449 " GRPKNGCIV 277 Epitope " AQHPNAELL 278 mimic of natural MuLV 75-83 " CCLCLTVFL 301 gag75K

P. CSP 375-383 p290 YENDIEKK 315 Falci arum " " 371-379 " DELDYENDI 315 HIV -1RT 206-214 " TEMEKEGKI 316 Rabies NS 197-205 VEAEIAH I 309, 310 InfluenzaNS1 152-160 " EEGANGEI 304 A

Murine SMCY 291 TENSGKDI 317 MHC class3-11 p293 AMAPRTLLL 318 1 leader NDlal 1-12 293 FFINILTLLVP 323 ha ND Beta 1-12 293 FFINIZTLLVP 323 ND al 1-17 " FFINILTLLVPILIAM324 ha ND Beta 1-17 " FFINALTLLVPILIAM"

COI 1-6 " FINRW 325 mitochondri al L. LemA 1-6 " IGWII 326 monocytoge nes SN gag 179-190 Mamu-A*O1EGCTPYDINQML 334 5,554,506 " IMPKAGLLI "

" KIWEELSVL "

" ALVETSYVKV "

" ThrLeuValGluValThrL"

euGl GIuVaI

" AlaLeuSerArgLysValA"

laGIuLeu " IleMetProLysAlaGIyLe"

uLeuIle " LysIleTrpGluGluLeuSe"

rValLeu " AlaLeuVaIGIuThrSerT"

yrValLysVa1 peptides HLA-A2 Lys Gly Ile 5,989,565 Leu Gly which Phe Val Phe bind Thr Leu to MHCs Thr Val " Gly Ile Ile "
Gly Phe Val Phe Thr Ile " Gly Ile Ile "
Gly Phe Val Phe Thr Leu " Gly Ile Leu "
Gly Phe Val Phe Thr Leu " Gly Leu Leu "
Gly Phe Val Phe Thr Leu " XXTVXXGVX, "

X=Leu or Ile " Ile Leu Thr "
Val Ile Leu Gl Val Leu " Tyr Leu Glu "
Pro Gly Pro Val Thr Ala " Gln Val Pro "
Leu Arg Pro Met Thr T r L s " Asp Gly Leu "
Ala Pro Pro Gln His Leu Ile Arg " Leu Leu Gly "
Arg Asn Ser Phe Glu Val Peptides HLA-C GluHisSerAlaTyrGlyG5,558,995 from clone luProArgLysLeuLeuTh MAGE-1 rGlnAs Leu " GluHisSerAlaTyrGlyG"

luProAr LysLeuLeu " SerAlaTyrGlyGluProA"

rgLysLeu GAGE HLA-Cw6 TyrArgProArgProArgA5,648,226 rgTyr " ThrTyrArgProArgProA"

rgArgT r " TyrArgProArgProArgA"

rgTyrV al " ThrTyrArgProArgProA"

r Ar T Val " ArgProArgProArgArgT"

rValGlu " MetSerTrpArgGlyArgS"

erThrTyrArgProArgPro ~g~g " ThrTyrArgProArgProA"

rgArgTyrV alGluProPro GluMetIle MAGE HLA-A1, Isolated nonapeptide5,405,940 primarilyhaving Glu at its N

terminal, Tyr at its C-terminal, and Asp at the third residue from its N terminal, with the proviso that said isolated nonapeptide is not Glu Ala Asp Pro Thr Gly His Ser Tyr (SEQ ID NO:
1 ), and wherein said isolated nonapeptide binds to a human leukocyte antigen molecule on a cell to form a complex, said complex rovokin 1 sis of said cell by a cytolytic T

cell specific to said complex " GluValValProIleSerHis"

LeuT r " GluValValArgIleGlyHi"

sLeuT r " GluValAspProIleGlyHi"

sLeuT

" GluValAspProAlaSerA"

snThrTyr " GluValAspProThrSerA"

snThrT r " GluAlaAspProThrSerA"

snThrTyr " GluValAspProIleGlyHi"

sValTyr " GAAGTGGTCCCCAT "

CAGCCACTTGTAC

" GAAGTGGTCCGCA "

TCGGCCACTTGTAC

" GAAGTGGACCCCA "

TCGGCCACTTGTAC

" GAAGTGGACCCCG "

CCAGCAACACCTAC

" GAAGTGGACCCCA "

CCAGCAACACCTAC

" GAAGCGGACCCCA "

CCAGCAACACCTAC

" GAAGCGGACCCCA "

CCAGCAACACCTAC

" GAAGTGGACCCCA "

TCGGCCACGTGTAC

" GluAlaAspProThrGly"

HisSer " AlaAspProTrpGlyHisS"

erT

MAGE HLA-A2 SerThrLeuValGluValT5,554,724 a tides hrLeuGl GluVa1 " " LeuValGluValThrLeu"

Gl GIuVaI

" " LysMetValGluLeuVa1"

HisPheLeu " " ValIlePheSerLysAlaSe"

rGluT rLeu " " TyrLeuGlnLeuValPhe"

Gl IleGluVa1 " " GInLeuValPheGlyIleG1"

uValVal " " GInLeuValPheGlyIleG1"

uValValGluVal " " IleIleValLeuAlaIleIleA"

laIle " " LysIleTrpGluGluLeuSe"

rMetLeuGluVal " " AlaLeuIleGluThrSerTy"

rValLysVal " " LeuIleGluThrSerTyrVa"

ILysVal " " GlyLeuGluAlaArgGly"

GluAlaLeuG1 Leu " " GlyLeuGIuAlaArgGly"

GIuAlaLeu " " AlaLeuGlyLeuValGly"

AlaGlnAla " " GIyLeuValGlyAlaGln"

AIaProAla " " AspLeuGluSerGluPhe"

GlnAlaAla " " AspLeuGluSerGluPhe"

GlnAlaAlaIle " " AlaIleSerArgLysMetV"

alGluLeuVa1 " " AlaIleSerArgLysMetV"

aIGIuLeu " " LysMetValGluLeuVa1"

HisPheLeuLeu " " LysMetValGluLeuVal"

HisPheLeuLeuLeu " " LeuLeuLeuLysTyrArg"

AlaAr GluProVa1 " " LeuLeuLysTyrArgAla"

Ar GluProVa1 " " ValLeuArgAsnCysGln"

As PhePheProVal " " TyrLeuGInLeuValPhe"

Gl IleGluValVa1 " " GlyIleGluValValGluV"

aIVaIProIle " " ProIleSerHisLeuTyrIle"

LeuVal " " HisLeuTyrIleLeuValTh"

rC sLeu " " HisLeuTyrIleLeuValTh"

rC sLeuGl Leu " " TyrIleLeuValThrCysLe"

uGl Leu " " CysLeuGlyLeuSerTyr"

As Gl Leu " " CysLeuGlyLeuSerTyr"

As G1 LeuLeu " " ValMetProLysThrGIyL"

euLeuIle " " ValMetProLysThrGIyL"

euLeuIlelle " " ValMetProLysThrGlyL"

euLeuIleIleVal " " GlyLeuLeuIleIleValLe"

uAlaIle " " GlyLeuLeuIleIleValLe"

uAlaIleIle " " GlyLeuLeuIleIleValLe"

uAlaIleIleAla " " LeuLeuIleIleValLeuA1"

aIleIle " " LeuLeuIleIleValLeuA1"

aIleIleAla " " LeuLeuIleIleValLeuA1"

aIleIleAlaIle " " - LeuIleIleValLeuAlaIleI"

leAla " " LeuIleIleValLeuAlaIleI"

leAlaIle ' " " IIeIleAlaIleGluGlyAsp"

C sAla " " LysIleTrpGluGluLeuSe"

rMetLeu " " LeuMetGlnAspLeuVa1"

GlnGluAsnT rLeu " " PheLeuTrpGlyProArg"

AlaLeuIle " " LeuIleGluThrSerTyrVa"

1L sVal " " AlaLeuIleGluThrSerTy"

rValL sValLeu " " ThrLeuLysIleGlyGlyG1"

uProHisIle " " HisIleSerTyrProProLeu"

HisGluAr Ala " " GlnThrAlaSerSerSerSe"

rThrLeu " " GlnThrAlaSerSerSerSe"

rThrLeuVal " " ValThrLeuGlyGluValP"

roAlaAla " " ValThrLysAlaGluMetL"

euGluSerVa1 " " ValThrLysAlaGluMetL"

euGluSerValLeu " " VaIThrCysLeuGlyLeu"

SerTyrAs G1 Leu " " LysThrGlyLeuLeuIleI1"

eValLeu " " LysThrGIyLeuLeuIleIl"

eValLeuAla " " LysThrGlyLeuLeuIleI1"

eValLeuAlaIle " " HisThrLeuLysIleGlyGl"

GluProHisIle " " MetLeuAspLeuGlnPro"

GluThrThr Mage-3 HLA-A2 GlyLeuGluAlaArgGly5,585,461 a tides GluAlaLeu " " AlaLeuSerArgLysValA"

IaGluLeu " " PheLeuTrpGlyProArg"

AlaLeuVal " " ThrLeuValGluValThrL"

euGl GluVa1 " " AlaLeuSerArgLysValA"

laGluLeuVal " " AlaLeuValGluThrSerT"

rValL sVal Tyrosinase HLA-A2 TyrMetAsnGlyThrMet5,487,974 SerGlnVal " " MetLeuLeuAlaValLeu"

T sLeuLeu Tyrosinase HLA-A2 MetLeuLeuAlaValLeu5,530,096 T rC sLeu " " LeuLeuAlaValLeuTyr"

C sLeuLeu Tyrosinase HLA-A2 SerGluIleTrpArgAspIle5,519,117 and HLA-B44 As PheAlaHisGluAla " " SerGluIleTrpArgAspIle"

As Phe " " GluGluAsnLeuLeuAsp"

PheValAr Phe Melan EAAGIGIL,TV Jager, E.
et al.

A/MART-1 Granulocyte-macrophage-colony-stimulating Factor Enhances Immune Responses To Melanoma-'associated Peptides in vivo hit. J Cancer 67, 54-62 1996) T rosinase MLLAVLYCL "

" YMDGTMS V "

gp100/Pmel YLEPGPVTA "

" LLDGTATLRL "

Influenza GILGFVFTL "

matrix MAGE-1 EADPTGHSY "

FROM

DAVID'S
LIST

BAGE HLA-C MAARAVFLALSAQL "

LQARLMKE

" " MAARAVFLALSAQL "

LQ

" " AARAVFLAL "

InfluenzaPR8 NP 147-154 Kd IYQRIRALV Falk et al., Allele-specific motifs revealed by sequencing of self peptides eluted from MHC molecules SELF P815 " SYFPEITHI "

PEPTIDE

InfluenzaJap HA " IYATVAGSL "

VYQILAIYA "

" " ~~ IYSTVASSL "

" JAP HA ~~ LYQNVGTYV "

HLA-A24 ~~ RYLENQKRT "

HLA-Cw3 " RYLKNGKET "

P815 " KYQAVTTTL "

PlasmodiumCSP " SYIPSAEKI "

berghen PlasmodiumCSP " SYVPSAFQI "

yoelli VesicularNP 52-59 Kb RGYVYQGL "

stomatitis viruse Ovalbumin " SIINFEKL "

Sandal NP 321-332 " APGNYPAL "

Virus VPYGSFKHV Morel et al., Processing of some antigens by the standard proteasome but not by the immunoproteaso me results in poor presentation by dendritic cells, Immunity, vol.

12:107-117, 2000.

MOTIFS

influenzaPR8 NP Kd restrictedTYQRTRALV 5,747,269 peptide motif self P815 " SYFPEITHI "
a tide influenzaJAP HA " IYATVAGSL "

influenzaJAP HA " VYQILAIYA "

influenzaPR8 HA " IYSTVASSL "

influenzaJAP HA " LYQNVGTYV "

HLA-A24 RYLENGKETL "

HLA-Cw3 RYLKNGKETL "

P815 " KYQAVTTTL "

tumour antigen PlasmodiumCSP " SYIPSAEKI "

ber hei PlasmodiumCSP ~ " SYVPSAEQI "

oeli influenzaNP Db - ASNENMETM "

restricted peptide motif adenovirusElA " SGPSNTPPEI "

lymphocytic " SGVENPGGYCL "

choriomenin gitis simian 40 T " SAINNY . . . "
virus HIV reverse HLA-A2.1-ILKEPVHGV "

transcriptase restricted peptide motif influenza " GILGFVFTL "

matrix rotein influenzainfluenza " ILGFVFTLTV "

matrix rotein HIV Ga rotein FL SRPEPT "

HIV Ga rotein AM MLKE . . "

HIV Ga rotein PIAPG MRE "

HIV Ga rotein MKDCTER "

HLA- VYGVIQK "

A*0205-restricted peptide motif Table S
VSV-NP a tide 49-62 LCMV-NP a tide 118-132 LCMV 1 co rotein a tide. 33-41 ISN LTLDSNTKYFHKLN
ISNQLTLDSNTKYFHKL
ISNQLTLDSNTKYFHK
VDTFLEDVKNLYHSEA
KPRAIVVDPVHGFMY
K TISPDYRNMI
Y DFIMDPKEKDKV

LLSFVRDLN YRADI
LPKPPKPV SKMRMATPL
LPKPPKPV SKMRMATPLLMQALP
LPKPPKPVSKMRMATPLLM ALPM
PKPPKP V SKMRMATPL
PKPPKPVSKMRMATPLLM A
KPPKPVSKMRMATPLLMQ
KPPKPVSKMRMATPLLMQALPM
VDDTQFVRFDSDAAS
ATKYGNMTEDHVMHLLQNA
VFLLLLADKVPETSLS
LNKILLDE A WK
GPPKLDIRKEEK IMIDIFH
GPPKLDIRKEEK IM>DIFHP
GFKAIRPDKKSNPIIRTV
YANILLDRRVP TDMTF
NLFLKSDGRIKYTLNKNSLK
IPDNLFLKSDGRIKYTLNKN
IPDNLFLKSDGRIKYTLNK
IPDNLFLKSDGRIKYTLN

NLFLKSDGRIKYTLNK
NLFLKSDGRIKYTLN
VTTLNSDLKYNALDLTN
VGSDWRFLRGYHQYA
[0078] Still further embodiments are directed to methods, uses, therapies and compositions related to epitopes with specificity for MHC, including, for example, those listed in Tables 6-10. Other embodiments include one or more of the MHCs listed in Tables 6-10, including combinations of the same, while other embodiments specifically exclude any one or more of the MHCs or combinations thereof. Tables 8-10 include frequencies for the listed HLA antigens.
Table 6 Class I MHC Molecules Class I
Human HLA-A*0101 HLA-A*0201 HLA-A*0202 HLA-A*0203 HLA-A*0204 HLA-A*0205 HLA-A*0206 HLA-A*0207 HLA-A*0209 HLA-A*0214 HLA-A*0301 HLA-A* 11 O 1 HLA-A*2902 HLA-A*3101 HLA-A*3302 HLA-A*6801 HLA-A*6901 HLA-B*0702 HLA-B*0703 HLA-B *0704 HLA-B*0705 HLA-B*1501 (B62) HLA-B *2702 HLA-B*2704 HLA-B*2705 HLA-B*2709 HLA-B*3501 HLA-B*3502 HLA-B*3701 HLA-B*3801 HLA-B*39011 HLA-B *3902 HLA-B*40012 (B60) HLA-B*4006 (B61) HLA-B*4402 HLA-B*4403 HLA-B*4501 HLA-B *4601 HLA-B*5101 HLA-B * 5102 HLA-B*5103 HLA-B*5201 HLA-B*5301 HLA-B*5401 HLA-B*5501 HLA-B*5502 HLA-B*5601 HLA-B*5801 HLA-B*6701 HLA-B*7301 HLA-B*7801 HLA-Cw*0102 HLA-Cw*0301 HLA-Cw*0304 HLA-Cw*0401 HLA-Cw *0601 HLA-Cw*0602 HLA-Cw*0702 HLA-Cw8 HLA-Cw*1601 M
HLA-G
Murine H2-Kd H2-Dd H2-Ld H2-Kb H2-Db H2-K'' H2-K'""' Qa-la Qa-2 Rat RT 1.Aa RT 1.A' Bovine Bota-Al l Bota-A20 Chicken Chimpanzee Patr-A*04 Patr-A* 11 Patr-B*01 Patr-B * 13 Patr-B * 16 Baboon Papa-A*06 Macaque Mamu-A*01 Swine SLA (haplotype d/d) Virus homolog hCMV class I homolog UL18 Table 7 Class I MHC Molecules Class I
Human HLA-Al HLA-A*0101 HLA-A*0201 HLA-A*0202 HLA-A*0204 HLA-A*0205 HLA-A*0206 HLA-A*0207 HLA-A*0214 HLA-A* 11 O 1 HLA-A*2902 HLA-A*3101 HLA-A*3302 HLA-A*6801 HLA-A*6901 HLA-B*0702 HLA-B *0703 HLA-B*0704 HLA-B *0705 HLA-B*1501 (B62) HLA-B *2702 HLA-B *2705 HLA-B*3501 HLA-B*3502 HLA-B*3701 HLA-B*3801 HLA-B*39011 HLA-B*3902 HLA-B*40012 (B60) HLA-B*4006 (B61) HLA-B*4402 HLA-B*4403 HLA-B *4641 HLA-B*5101 HLA-B*5102 HLA-B*5103 HLA-B*5201 HLA-B*5301 HLA-B * 5401 HLA-B*5501 HLA-B*5502 HLA-B*5601 HLA-B*5801 HLA-B *6701 HLA-B*7301 HLA-B*7801 HLA-Cw*0102 HLA-Cw*0301 HLA-Cw*0304 HLA-Cw*0401 HLA-Cw*0601 HLA-Cw*0602 HLA-Cw*0702 HLA-G
Murine H2-K'' H2-Dd H2-Ld H2-Kb H2-Db H2-Kk H2-K'""' Qa-2 Rat RTl .A~
RT1.A~
Bovine Bota-Al l Bota-A20 Chicken Virus homolog hCMV class I homolog UL18 Table 8 [0079) Estimated gene frequencies of HLA-A antigens CAU AFR ASI LAT NAT

Antigen G~ SEb Gf SE Gf SE Gf SE Gf SE

A1 15.18430.04895.72560.07714.48180.08467.40070.097812.03160.2533 A2 28.65350.061918.88490.131724.63520.179428.11980.170029.34080.3585 A3 13.38900.04638.44060.09252.64540.06558.07890.101911.02930.2437 A28 4.46520.02809.92690.09971.76570.05378.94460.10675.38560.1750 A36 0.02210.00201.88360.04480.01480.00490.15840.01480.15450.0303 A23 1.82870.018110.20860.10100.32560.02312.92690.06281.99030.1080 A24 9.32510.03952.96680.056022.03910.172213.26100.127112.66130.2590 A9 unsplit0.08090.00380.03670.00630.08580.01190.05370.00860.03560.0145 A9 total11.23470.042913.21210.112822.45050.173316.24160.138214.68720.2756 A25 2.11570.01950.43290.02160.09900.01281.19370.04041.45200.0924 A26 3.87950.02622.82840.05474.66280.08623.26120.06622.42920.1191 A34 0.15080.00523.52280.06101.35290.04700.49280.02600.31500.0432 A43 0.00180.00060.03340.00600.02310.00620.00550.00280.00590.0059 A66 0.01730.00180.22330.01550.04780.00890.03990.00740.05340.0178 A10 unsplit0.07900.00380.09390.01010.12550.01440.06470.00940.02980.0133 A10 total6.24410.03287.13480.08506.31110.09935.05780.08164.28530.1565 A29 3.57960.02523.20710.05821.12330.04294.51560.07743.43450.1410 A30 2.50670.021213.09690.11292.20250.05984.48730.07722.53140.1215 A31 2.73860.02211.65560.04203.60050.07614.83280.08006.08810.1855 A32 3.69560.02561.53840.04051.03310.04112.70640.06042.55210.1220 A33 1.20800.01486.56070.08229.27010.11912.65930.05991.07540.0796 A74 0.02770.00221.99490.04610.05610.00960.20270.01670.10680.0252 A19 unsplit0.05670.00320.20570.01490.09900.01280.12110.01290.04750.0168 A19 total13.81290.046828.25930.150417.38460.155519.52520.148115.83580.2832 AX 0.82040.02974.95060.09632.99160.11771.63320.08781.84540.1925 Gene frequency.
bStandard error.
Table 9 Estimated gene frequencies for HLA-B antigens CAU AFR ASI LAT NAT

AntigenG~ SE Gf SE Gf SE Gf SE Gf SE

B7 12.17820.044510.59600.10244.26910.08276.44770.091810.98450.2432 B8 9.40770.03973.83150.06341.33220.04673.82250.07158.57890.2176 B 13 2.30610.02030.81030.02954.92220.08861.26990.04161.74950.1013 B 14 4.34810.02773.03310.05660.50040.02875.41660.08462.98230.1316 B 18 4.79800.02903.20570.05821.12460.04294.23490.07523.34220.1391 B27 4.38310.02781.29180.03722.23550.06032.37240.05675.19700.1721 B35 9.66140.04028.51720.09278.12030.112214.65160.132910.11980.2345 B37 1.40320.01590.59160.02521.23270.04490.78070.03270.97550.0759 B41 0.92110.01290.81830.02960.13030.01471.28180.04180.47660.0531 B42 0.06080.00335.69910.07680.08410.01180.58660.02840.28560.0411 B46 0.00990.00130.01510.00404.92920.08860.02340.00570.02380.0119 B47 0.20690.00610.13050.01190.09560.01260.18320.01590.21390.0356 B48 0.08650.00400.13160.01192.02760.05751.59150.04661.02670.0778 B53 0.46200.009210.95290.10390.43150.02661.69820.04811.08040.0798 CAU AFR ASI LAT NAT

AntigenGf SE Gf SE Gf SE Gf SE Gf SE

B59 0.00200.00060.00320.00190.42770.02650.00550.00280' B67 0.00400.00090.00860.00300.22760.01940.00550.00280.00590.0059 B70 0.32700.00777.35710.08660.89010.03821.92660.05120.69010.0639 B73 0.01080.00140.00320.00190.01320.00470.02610.00600' B51 5.42150.03072.59800.05257.47510.10806.81470.09436.90770.1968 B52 0.96580.01321.37120.03833.51210.07522.24470.05520.69600.0641 B5 unsplit0.15650.00530.15220.01280.12880.01460.15460.01460.13070.0278 B5 total6.54380.04354.12140.074711.11600.15049.21410.13247.73440.2784 B44 13.48380.04657.01370.08475.68070.09489.92530.112111.80240.2511 B45 0.57710.01024.80690.07080.18160.01731.88120.05060.76030.0670 B12 0.07880.00380.02800.00550.00490.00290.01930.00510.06540.0197 unsplit B12 14.14400.047411.84860.10725.86730.096311.82580.121012.62810.2584 total 6.9421 B62 5.91170.03201.52670.04049.22490.11904.18250.0747 p,1973 0.3738 B63 0.43020.00881.88650.04480.44380.02700.80830.0333 p,p471 0.0356 B75 0.01040.00140.02260.00491.96730.05660.11010.0123 0.0145 p B76 0.00260.00070.00650.00260.08740.01200.00550.0028 B77 0.00570.00100.01190.00360.05770.00980.00830.0034 0.0059 0.0059 B 15 0.13050.00490.06910.00860.43010.02660.18200.0158 p,p206 unsplit 0.0715 B15 6.49100.03343.52320.060812.21120.13445.29670.0835 p,2035 total 7.4290 B38 2.44130.02090.33230.01893.28180.07281.96520.05171.10170.0806 B39 1.96140.01881.28930.03712.03520.05766.30400.09094.55270.1615 B16 0.06380.00340.02370.00510.06440.01030.12260.01300.05930.0188 unsplit B16 4.46670.02801.64530.04195.38140.09218.39170.10365.71370.1797 total B57 3.59550.02525.67460.07662.57820.06472.18000.05442.72650.1260 B58 0.71520.01145.95460.07844.01890.08031.24810.04130.93980.0745 B 17 0.28450.00720.32480.01870.37510.02480.14460.01410.26740.0398 unsplit B17 4.59520.028411.95400.10766.97220.10413.57270.06913.93380.1503 total B49 1.64520.01722.62860.05280.24400.02002.33530.05621.54620.0953 B50 1.05800.01380.86360.03040.44210.02701.88830.05070.78620.0681 B21 0.07020.00360.02700.00540.01320.00470.07710.01030.03560.0145 unsplit B21 2.77330.02223.51920.06080.69930.03394.30070.07552.36800.1174 total B54 0.01240.00150.01830.00442.68730.06600.02890.00630.05340.0178 B55 1.90460.01850.48950.02292.24440.06040.95150.03611.40540.0909 B56 0.55270.01000.26860.01700.82600.03680.35960.02220.33870.0448 B22 0.16820.00550.04960.00730.27300.02120.03720.00710.12460.0272 unsplit B22 2.08520.02170.82610.02976.03070.09711.37710.04331.92210.1060 total B60 5.22220.03021.52990.04048.32540.11352.25380.05535.72180.1801 B61 1.19160.01470.47090.02256.20720.09894.66910.07882.60230.1231 B40 0.26960.00700.03880.00650.32050.02300.24730.01840.22710.0367 unsplit B40 6.68340.03382.03960.046514.85310.14627.17020.09638.55120.2168 total BX 1.09220.02523.52580.08023.87490.09882.52660.0807( 1.98670.1634 ~

Gene frequency. bStandard error. °The observed gene count was zero.

Table 10.
Estimated gene frequencies of HLA-DR antigens CAU AFR ASI LAT NAT

Antigen G~ SEb Gf Gf SE Gf SE Gf SE
SE

DR1 10.22790.04136.82000.08323.46280.07477.98590.10138.25120.2139 DR2 15.24080.049116.23730.122218.61620.160811.23890.118215.39320.2818 DR3 10.87080.042413.30800.11244.72230.08677.89980.100810.25490.2361 DR4 16.75890.05115.70840.076515.46230.149020.53730.152019.82640.3123 DR6 14.39370.047918.61170.129113.44710.140417.02650.141114.80210.2772 DR7 13.28070.046310.13170.09976.92700.104010.67260.11 10.42190.2378 DR8 2.88200.02276.26730.08006.54130.10139.77310.11106.00590.1844 DR9 1.06160.01392.96460.05599.75270.12181.07120.03832.86620.1291 DR10 1.47900.01632.03970.04652.23040.06021.80440.04951.08960.0801 DR11 9.31800.039610.61510.10184.73750.08697.04110.09555.31520.1740 DR12 1.90700.01854.11520.065510.13650.12391.72440.04842.01320.1086 DR5 unsplit1.21990.01492.29570.04931.41180.04801.82250.04981.67690.0992 DR5 total12.44490.004517.02600.124316.28580.151610.58800.11489.00520.2218 DRX 1.35980.03420.88530.07602.55210.10891.40230.09302.08340.2037 ''Gene frequency.
bStandard error.
[0080] It can be desirable to express housekeeping peptides in the context of a larger protein. Processing can be detected even when a small number of amino acids are present beyond the terminus of an epitope. Small peptide hormones are usually proteolytically processed from longer translation products, often in the size range of approximately 60-120 amino acids. This fact has led some to assume that this is the minimum size that can be efficiently translated. In some embodiments, the housekeeping peptide can be embedded in a translation product of at least about 60 amino acids, in others 70, 80, 90 amino acids, and in still others 100, 110 or 120 amino acids, for example. In other embodiments the housekeeping peptide can be embedded in a translation product of at least about 50, 30, or 15 amino acids.
[0081] Due to differential proteasomal processing, the immunoproteasome of the pAPC
produces peptides that are different from those produced by the housekeeping proteasome in peripheral body cells. Thus, in expressing a housekeeping peptide in the context of a larger protein, it is preferably expressed in the pAPC in a context other than its full-length native sequence, because, as a housekeeping epitope, it is generally only efficiently processed from the native protein by the housekeeping proteasome, which is not active in the pAPC. In order to encode the housekeeping epitope in a DNA sequence encoding a larger polypeptide, it is useful to find flanking areas on either side of the sequence encoding the epitope that permit appropriate cleavage by the immunoproteasome in order to liberate that housekeeping epitope. Such a sequence promoting appropriate processing is referred to hereinafter as having substrate or liberation sequence function.
Altering flanking amino acid residues at the N-terminus and C-terminus of the desired housekeeping epitope can facilitate appropriate cleavage and generation of the housekeeping epitope in the pAPC.
Sequences embedding housekeeping epitopes can be designed de novo and screened to determine which can be successfully processed by immunoproteasomes to liberate housekeeping epitopes.
[0082] Alternatively, another strategy is very effective for identifying sequences allowing production of housekeeping epitopes in APC. A contiguous sequence of amino acids can be generated from head to tail arrangement of one or more housekeeping epitopes.
A construct expressing this sequence is used to immunize an animal, and the resulting T
cell response is evaluated to determine its specificity to one or more of the epitopes in the array. These immune responses indicate housekeeping epitopes that are processed in the pAPC
effectively. The necessary flanking areas around this epitope are thereby defined. The use of flanking regions of about 4-6 amino acids on either side of the desired peptide can provide the necessary information to facilitate proteasome processing of the housekeeping epitope by the immunoproteasome.
Therefore, a substrate or liberation sequence of approximately 16-22 amino acids can be inserted into, or fused to, any protein sequence effectively to result in that housekeeping epitope being produced in an APC. In some embodiments, a broader context of a substrate sequence can also influence processing. In such embodiments, comparisons of a liberaton sequence in a variety of contexts can be useful in further optimizing a particular substrate sequence. In alternate embodiments the whole head-to-tail array of epitopes, or just the epitopes immediately adjacent to the correctly processed housekeeping epitope can be similarly transferred from a test construct to a vaccine vector.
[0083] In a preferred embodiment, the housekeeping epitopes can be embedded between known immune epitopes, or segments of such, thereby providing an appropriate context for processing. The abutment of housekeeping and immune epitopes can generate the necessary context to enable the immunoproteasome to liberate the housekeeping epitope, or a larger fragment, preferably including a correct C-terminus. It can be useful to screen constructs to verify that the desired epitope is produced. The abutment of housekeeping epitopes can generate a site cleavable by the immunoproteasome. Some embodiments of the invention employ known epitopes to flank housekeeping epitopes in test substrates; in others, screening as described below is used, whether the flanking regions are arbitrary sequences or mutants of the natural flanking sequence, and whether or not knowledge of proteasomal cleavage preferences are used in designing the substrates.
[0084] Cleavage at the mature N-terminus of the epitope, while advantageous, is not required, since a variety of N-terminal trimming activities exist in the cell that can generate the mature N-terminus of the epitope subsequent to proteasomal processing. It is preferred that such N-terminal extension be less than about 25 amino acids in length and it is further preferred that the extension have few or no proline residues. Preferably, in screening, consideration is given not only to cleavage at the ends of the epitope (or at least at its C-terminus), but consideration also can be given to ensure limited cleavage within the epitope.
[0085] Shotgun approaches can be used in designing test substrates and can increase the efficiency of screening. In one embodiment multiple epitopes can be assembled one after the other, with individual epitopes possibly appearing more than once. The substrate can be screened to determine which epitopes can be produced. In the case where a particular epitope is of concern, a substrate can be designed in which it appears in multiple different contexts.
When a single epitope appearing in more than one context is liberated from the substrate additional secondary test substrates, in which individual instances of the epitope are removed, disabled, or are unique, can be used to determine which are being liberated and truly confer substrate or liberation sequence function.
[0086] Several readily practicable screens exist. A preferred in vitro screen utilizes proteasomal digestion analysis, using purified immunoproteasomes, to determine if the desired housekeeping epitope can be liberated from a synthetic peptide embodying the sequence in question.
The position of the cleavages obtained can be determined by techniques such as mass spectrometry, HPLC, and N-terminal pool sequencing; as described in greater detail in U.S.
Patent Application Nos. 09/561,074, 09/560,465 and 10/117,937, and Provisional U.S. Patent Application Nos.
60/282,211, 60/337,017, and 60/363, 210.
[0087] Alternatively, in vivo and cell-based screens such as immunization or target sensitization can be employed. For immunization a nucleic acid construct capable of expressing the sequence in question is used. Harvested CTL can be tested for their ability to recognize target cells presenting the housekeeping epitope in question. Such targets cells are most readily obtained by pulsing cells expressing the appropriate MHC molecule with synthetic peptide embodying the mature housekeeping epitope. Alternatively, immunization can be carried out using cells known to express housekeeping proteasome and the antigen from which the housekeeping epitope is derived, either endogenously or through genetic engineering. To use target sensitization as a screen, CTL, or preferably a CTL clone, that recognizes the housekeeping epitope can be used.
In this case it is the target cell that expresses the embedded housekeeping epitope (instead of the pAPC during immunization) and it must express immunoproteasome. Generally, the cell or target cell can be transformed with an appropriate nucleic acid construct to confer expression of the embedded housekeeping epitope. Loading with a synthetic peptide embodying the embedded epitope using peptide loaded liposomes, or complexed with cationic lipid protein transfer reagents such as BIOPORTERTM (Gene Therapy Systems, San Diego, CA), represents an alternative.
[0088] Once sequences with substrate or liberation sequence function are identified they can be encoded in nucleic acid vectors, chemically synthesized, or produced recombinantly. In any of these forms they can be incorporated into immunogenic compositions. Such compositions can be used in vitro in vaccine development or in the generation or expansion of CTL
to be used in adoptive immunotherapy. In vivo they can be used to induce, amplify or sustain and active immune response.
The uptake of polypeptides for processing and presentation can be greatly enhanced by packaging with cationic lipid, the addition of a tract of cationic amino acids such as poly-L-lysine (Ryser, H.J.
et al., J. Cell Physiol. 113:167-178, 1982; Shen, W.C. & Ryser, H.J., Proc.
Natl. Aced. Sci. USA
75:1872-1876, 1978), the incorporation into branched structures with importation signals (Sheldon, K. et al., Proc. Natl. Aced. Sci. USA 92:2056-2060, 1995), or mixture with or fusion to polypeptides with protein transfer function including peptide earners such as pep-1 (Morris, M.C., et al., Nat.
Biotech. 19:1173-1176, 2001), the PreS2 translocation motif of hepatitis B
virus surface antigen, VP22 of herpes viruses, and HIV-TAT protein (Oess, S. & Hildt, E., Gene Ther.
7:750-758, 2000;
Ford, K.G., et al., Gene Ther. 8:1-4, 2001; Hung, C.F. et al., J. virol.
76:2676-2682, 2002; Oliveira, S.C., et a;. Hum. Gene Ther. 12:1353-1359, 2001; Normand, N. et al., J. Biol.
Chem. 276:15042-15050, 2001; Schwartz, J.J. & Zhang, S., Curr. Opin. Mol. Ther. 2:162-167, 2000; Elliot G., 7 Hare, P. Cell 88:223-233, 1997), among other methodologies. Particularly for fusion proteins the immunogen can be produced in culture and the purified protein administered or, in the alternative, the nucleic acid vector can be administered so that the immunogen is produced and secreted by cells transformed in vivo. In either scenario the transport function of the fusion protein facilitates uptake by pAPC.
EXAMPLES
Example 1 [0089] A recombinant DNA plasmid vaccine, pMA2M, which encodes one polypeptide with an HLA A2-specific CTL epitope ELAGIGIL,TV (SEQ ID NO. 1) from melan-A
(26-35A27L), and a portion (amino acids 31-96) of melan-A (SEQ ID NO. 2) including the epitope clusters at amino acids 31-48 and 56-69, was constructed. These clusters were previously disclosed in U.S.
Patent Application No. 09/561,571 entitled EPITOPE CLUSTERS. Flanking the defined melan-A
CTL epitope are short amino acid sequences derived from human tyrosinase (SEQ
ID NO. 3) to facilitate liberation of the melan-A housekeeping epitope by processing by the immunoproteasome.

In addition, these amino acid sequences represent potential CTL epitopes themselves. The cDNA
sequence for the polypeptide in the plasmid is under the control of promoter/enhancer sequence from cytomegalovirus (CMVp) (see Figure 1), which allows efficient transcription of messenger for the polypeptide upon uptake by APCs. The bovine growth hormone polyadenylation signal (BGH
polyA) at the 3' end of the encoding sequence provides a signal for polyadenylation of the messenger to increase its stability as well as for translocation out of nucleus into the cytoplasm for translation.
To facilitate plasmid transport into the nucleus after uptake, a nuclear import sequence (I~IIS) from simian virus 40 (SV40) has been inserted in the plasmid backbone. The plasmid carries two copies of a CpG immunostimulatory motif, one in the NIS sequence and one in the plasmid backbone.
Lastly, two prokaryotic genetic elements in the plasmid are responsible for amplification in E.coli, the kanamycin resistance gene (Kan R) and the pMB 1 bacterial origin of replication.
SUBSTRATE or LIBERATION sequence [0090] The amino acid sequence of the encoded polypeptide (94 amino acid residues in length) (SEQ ID NO. 4) containing a 28 amino acid substrate or liberation sequence at its N-terminus (SEQ ID NO. 5) is given below:
[0091] MLLAVLYCL-ELAGIGILTV-YMDGTMSQV-GILTVILGVLLLIGCWYCRRRNGYRALMDKSLHVGTQCALTRRCPQEGFDHRDSKVSLQEK
NCEPV
[0092] The first 9 amino acid residues are derived from tyrosinase~_9 (SEQ ID
NO. 6), the next ten constitute melan-A (26-35A27L) (SEQ )D NO. 1), and amino acid residues 20 to 29 are derived from tyrosinase369-377 (SEQ >D NO. 7). These two tyrosinase nonamer sequences both represent potential HLA A2-specific CTL epitopes. Amino acid residues 10-19 constitute melan-A
(26-35A27L) an analog of an HLA A2-specific CTL epitope from melan-A, EAAGIGILTV (SEQ ID
NO. 8), with an elevated potency in inducing CTL responses during in vitro immunization of human PBMC and in vivo immunization in mice. The segment of melan-A constituting the rest of the polypeptide (amino acid residues 30 to 94) contain a number of predicted HLA
A2-specific epitopes, including the epitope clusters cited above, and thus can be useful in generating a response to immune epitopes as described at length in the patent applications 'Epitope Synchronization in Antigen Presenting Cells' and 'Epitope Clusters'. This region was also included to overcome any difficulties that can be associated with the expression of shorter sequences. A drawing of pMA2M is shown in Figure 1.
Plasmid construction [0093] A pair of long complementary oligonucleotides was synthesized which encoded the first 30 amino acid residues. In addition, upon annealing, these oligonucleotides generated the cohensive ends of Afl II at the 5' end and that of EcoR I at the 3' end. The melan A3,_96 region was amplified with PCR using oligonucleotides carrying restriction sites for EcoR
I at the 5' end and Not I at the 3' end. The PCR product was digested with EcoR I and Not I and ligated into the vector backbone, described in Example 1, that had been digested with Afl II and Not I, along with the annealed oligonucleotides encoding the amino terminal region in a three-fragment ligation. The entire coding sequence was verified by DNA sequencing. The sequence of the entire insert, from the Afl II site at the 5' end to the Not I site at the 3' end is disclosed as SEQ
ID NO. 9. Nucleotides 12-293 encode the polypeptide.
Example 2 [0094] Three vectors containing melan-A (26-35A27L) (SEQ B7 NO. 1) as an embedded housekeeping epitope were tested for their ability to induce a CTL
response to this epitope in HLA-A2 transgenic HHD mice (Pascolo et al. J. Exp. Med. 185:2043-2051, 1997). One of the vectors was pMA2M described above (called pVAXM3 in Figure 3). In pVAXM2 the same basic group of 3 epitopes was repeated several times with the flanking epitopes truncated by differing degrees in the various repeats of the array. Specifically the cassette consisted o~
[0095] M-Tyr(5-9)-ELA-Tyr(369-373)-Tyr(4-9)-ELA-Tyr(369-374)-Tyr(3-9)-ELA-Tyr(369-375)-Tyr(2-9)-ELA
(SEQ ID NO. 10) [0096] where ELA represents melan-A (26-35A27L) (SEQ >D NO. 1). This cassette was inserted in the same plasmid backbone as used for pVAXM3. The third, pVAXMI is identical to pVAXM2 except that the epitope array is followed by an IRES (internal ribosome entry site for encephalomyocarditis virus) linked to a reading frame encoding melan-A 31-70.
[0097] Four groups of three HHD A2.1 mice were injected intranodally in surgically exposed inguinal lymph nodes with 25 pl of 1 mg/ml plasmid DNA in PBS on days 0, 3, and 6, each group receiving one of the three vectors or PBS alone. On day 14 the spleens were harvested and restimulated in vitro one time with 3-day LPS blasts pulsed with peptide (melan-A (26-35A27L)(SEQ ID NO. 1)). The in vitro cultures were supplemented with Rat T-Stim (Collaborative Biomedical Products) on the 3'd day and assayed for cytolytic activity on the 7'" day using a standard 51 Cr-release assay. Figures 2 to 5 show % specific lysis obtained using the cells immunized with PBS, pVAXMl, pVAXM2, and pVAXM3, respectively on T2 target cells and T2 target cells pulsed with melan-A (26-35A27L) (ELA) (SEQ ID NO. 1). All three vectors generated strong CTL
responses. These data indicated that the plasmids have been taken up by APCs, the encoded polypeptide has been synthesized and proteolytically processed to produce the decamer epitope in question (that is, it had substrate or liberation sequence function), and that the epitope became HLA-A2 bound for presentation. Also, an isolated variant of pVAXM2, that terminates after the 55'"
amino acid, worked similarly well as the full length version (data not shown).
Whether other potential epitopes within the expression cassette can also be produced and be active in inducing CTL
responses can be determined by testing for CTL activity against target cells pulsed with corresponding synthetic peptides.
Example 3 An NY-ESO-1 (SEQ ID NO. 11 ) SUBSTRATE/LIBERATION Sequence [0098] Six other epitope arrays were tested leading to the identification of a substrate/liberation sequence for the housekeeping epitope NY-ESO-1,57_165 (SEQ 117 NO. 12). The component epitopes of the arrays were:
[0099]SSX-24~-49: KASEKIFYV (SEQ ID NO. Array element 13) A

[O100JNY-ESO-1157-165SLLMWITQC (SEQ ID NO. Array element 12) B

[0101]NY-ESO-1 ~ TQCFLPVFL (SEQ m NO. Array element 63_ ~ ~ ~ 14) C
:

[0102]PSMA2gg_297: GLPS1PVHPI(SEQ ID NO. Array element 15) D

[0103]TYR4_9: AVLYCL (SEQ ID NO. Array element 16) E

[0104]The six arraysthe following had arrangements of elements after starting with an initiator methionine:
[OlOSJpVAX-PC-A: B-A-D-D-A-B-A-A

[0106]pVAX-PC-B: D-A-B-A-A-D-B-A

[0107)pVAX-PC-C: E-A-D-B-A-B-E-A-A

[0108]pVAX-BC-A: B-A-C-B-A-A-C-A

[0109)pVAX-BC-B: C-A-B-C-A-A-B-A

[0110] pVAX-BC-C: E-A-A-B-C-B-A-A
[0111] These arrays were inserted into the same vector backbone described in the examples above. The plasmid vectors were used to immunize mice essentially as described in Example 2 and the resulting CTL were tested for their ability to specifically lyse target cells pulsed with the peptide NY-ESO-1 157-165, corresponding to element B above. Both pVAX-PC-A and pVAX-BC-A were found to induce specific lytic activity. Comparing the contexts of the epitope (element B) in the various arrays, and particularly between pVAX-PC-A and pVAX-BC-A, between pVAX-PC-A and pVAX-PC-B, and between pVAX-BC-A and pVAX-BC-C, it was concluded that it was the first occurrence of the epitope in pVAX-PC-A and pVAX-BC-A that was being correctly processed and presented. In other words an initiator methionine followed by elements B-A constitute a substrate/liberation sequence for the presentation of element B. On this basis a new expression cassette for use as a vaccine was constructed encoding the following elements:
[0112] An initiator methionine, [0113] NY-ESO-1 ~ 57-165 (bold) - a housekeeping epitope, [0114] SSX24~-49 (italic) -providing appropriate context for processing, and [0115] NY-ESO-1 ~~_ ~ gp - to avoid "short sequence" problems and provide immune epitopes.
[0116) Thus the construct encodes the amino acid sequence:
[0117] M-SLLMWITQC-KASEKIFYV
RCGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPLPVPGVLLKEFTVSGNIL,TIRLTA
ADHRQLQLSISSCLQQLSLLMWITQCFLPVFLAQPPSGQRR (SEQ ID NO. 17) and MSLLMWITQCKASEKIFYV (SEQ ID NO. 18) constitutes the liberation or substrate sequence. A
polynucleotide encoding SEQ ID NO. 17 (SEQ ID NO. 19: nucleotides 12-380) was inserted into the same plasmid backbone as used for pMA2M generating the plasmid pN157.
Example 4 [0118] A construct similar to pN157 containing the whole epitope array from pVAX-PC-A was also made and designated pBPL. Thus the encoded amino acid sequence in pBPL is:
[0119] M-SLLMWITQC-KASEKIFYY GLPSIPVHPI-GLPSIPVHPI-KASEKIFYV
SLLMWITQC-KASEKIFYV KASEKIFYV
RCGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPLPVPGVLLKEFTVSGNIL,TIRLTA
ADHRQLQLSISSCLQQLSLLMWITQCFLPVFLAQPPSGQRR (SEQ m NO. 20).
[0120] SEQ LD NO. 21 is the polynucleotide encoding SEQ ID NO. 20 used in pBPL.

[0121] A portion of SEQ ID NO. 20, IKASEKIFYVSLLMWITQCKASEKIFYVK (SEQ
m NO. 22) was made as a synthetic peptide and subjected to in vitro proteasomal digestion analysis with human immunoproteasome, utilizing both mass spectrometry and N-terminal pool sequencing.
The identification of a cleavage after the C residue indicates that this segment of the construct can function as a substrate or liberation sequence for NY-ESO-1 ~5~_~65 (SEQ ID
NO. 12) epitope (see Figure 6). Figure 7 shows the differential processing of the SLLMWITQC epitope (SEQ ID NO. 12) in its native context where the cleavage following the C is more efficiently produced by housekeeping than immunoproteasome. The immunoproteasome also produces a major cleavage internal to the epitope, between the T and the Q when the epitope is in its native context, but not in the context of SEQ ID NO. 22 (compare fig. 6 and 7).
Example 5 [0122] Screening of further epitope arrays led to the identification of constructs promoting the expression of the epitope SSX-24_49 (SEQ ID NO. 13). In addition to some of the array elements defined in Example 3, the following additional elements were also used:
[0123) SSX-457-65:VMTKLGFKV (SEQ lD NO. 23) Array element F.
[0124] PSMA730-739 RQ~~TV (SEQ ID NO. 24) Array element G.
[0125] A construct, denoted CTLA02, encoding an initiator methionine and the.
array F-A-G-D-C-F-G-A, was found to successfully immunize HLA-A2 transgenic mice to generate a CTL
response recognizing the peptide SSX-241-49 (SEQ 117 NO. 13).
[0126] As described above, it can be desirable to combine a sequence with substrate or liberation sequence function with one that can be processed into immune epitopes. Thus SSX-2 ~ 5_ 183 (SEQ ~ NO. 25) was combined with all or part of the array as follows:
[0127] CTLS 1: F-A-G-D-C-F-G-A- SSX-2 ~ 5_ ~ g3 (SEQ 1D NO. 26) [0128] CTLS2: SSX-2 ~ 5_ ~ 83 - F-A-G-D-C-F-G-A (SEQ ID NO. 27) [0129] CTLS3: F-A-G-D- SSX-215-~ 83 (SEQ ID NO. 28) [0130] CTLS4: SSX-2~5_~g3 -C-F-G-A (SEQ ID NO. 29).
[0131] All of the constructs except CTLS3 were able to induce CTL recognizing the peptide SSX-24_49 (SEQ >D NO. 13). CTLS3 was the only one of these four constructs which did not include the second element A from CTLA02 suggesting that it was this second occurrence of the element that provided substrate or liberation sequence function. In CTLS2 and CTLS4.the A element is at the C-terminal end of the array, as in CTLA02. In CTLS1 the A element is immediately followed by the SSX-2 ~ 5_ ~ g3 segment which begins with an alanine, a residue often found after proteasomal cleavage sites (Toes, R.E.M., et al., J. Exp. Med. 194:1-12, 2001). SEQ )D NO. 30 is the polynucleotide sequence encoding SEQ ID NO. 26 used in CTLS I, also called pCBP.
(0132] A portion of CTLS1 (SEQ ID NO. 26), encompassing array elements F-A-SSX-215-23 with the sequence RQIYVAAFTV-KASEKIFYY AQIPEKIQK (SEQ ID NO. 31 ), was made as a synthetic peptide and subjected to in vitro proteasomal digestion analysis with human immunoproteasome, utilizing both mass spectrometry and N-terminal pool sequencing. The observation that the C-terminus of the SSX-241-49 epitope (SEQ ID NO. 13) was generated (see Figure 8) provided further evidence in support of substrate or liberation sequence function. The data in Figure 9 showed the differential processing of the SSX-24~-49 epitope, KASEKIFYh (SEQ >D NO.
13), in its native context, where the cleavage following the V was the predominant cleavage produced by housekeeping proteasome, while the immunoproteasome had several major cleavage sites elsewhere in the sequence. By moving this epitope into the context provided by SEQ >D NO. 31 the desired cleavage became a major one and its relative frequency compared to other immunoproteasome cleavages was increased (compare figs. 8 and 9). The data in figure 8B also showed the similarity in specificity of mouse and human immunoproteasome lending support to the usefulness of the transgenic mouse model to predict human antigen processing.
Example 6 [0133] Screening also revealed substrate or liberation sequence function for a tyrosinase epitope, Tyr2o~-215 (SEQ >D NO. 32), as part of an array consisting of the sequence [Tyre _ ~ 7- Tyr2o7-215]4~ ~MLLAVLYCLLWSFQTSA-FLPWHRLFL]4, (SEQ ID NO. 33). The same vector backbone described above was used to express this array. This array differs from those of the other examples in that the Tyr~_~7 segment, which was included as a source of immune epitopes, is used as a repeated element of the array. This is in contrast with the pattern shown in the other examples where sequence included as a source of immune epitopes and/or length occurred a single time at the beginning or end of the array, the remainder of which was made up of individual epitopes or shorter sequences.

Plasmid construction [0134] The polynucleotide encoding SEQ ID NO. 33 was generated by assembly of annealed synthetic oligonucleotides. Four pairs of complementary oligonucleotides were synthesized which span the entire coding sequence with cohesive ends of the restriction sites of Afl II and EcoR I
at either terminus. Each complementary pair of oligonucleotides were first annealed, the resultant DNA fragments were ligated stepwise, and the assembled DNA fragment was inserted into the same vector backbone described above pre-digested with Afl II/EcoR I. The construct was called CTLT2/pMEL and SEQ 117 NO. 34 is the polynucleotide sequence used to encode SEQ 117 NO. 33.
Example 7 Administration of a DNA ~lasmid formulation of a immunotherapeutic for melanoma to humans.
[0135] An MA2M melanoma vaccine with a sequence as described in Example 1 above, was formulated in 1 % Benzyl alcohol, I % ethyl alcohol, O.SmM EDTA, citrate-phosphate, pH 7.6.
Aliquots of 200, 400, and 600 pg DNA/ml were prepared for loading into MIhIIMED 407C infusion pumps. The catheter of a SILHOUETTE infusion set was placed into an inguinal lymph node visualized by ultrasound imaging. The pump and infusion set assembly was originally designed for the delivery of insulin to diabetics. The usual l7mm catheter was substituted with a 3lmm catheter for this application. The infizsion set was kept patent for 4 days (approximately 96 hours) with an infusion rate of about 25 pl/hour resulting in a total infused volume of approximately 2.4 ml. Thus the total administered dose per infusion was approximately 500, and 1000 pg;
and can be 1500 pg, respectively, for the three concentrations described above. Following an infusion, subjects were given a 10 day rest period before starting a subsequent infusion. Given the continued residency of plasmid DNA in the lymph node after administration and the usual kinetics of CTL response following disappearance of antigen, this schedule will be sufficient to maintain the immunologic CTL response.
Example 8 [0136] SEQ 1D NO. 22 is made as a synthetic peptide and packaged with a cationic lipid protein transfer reagent. The composition is infused directly into the inguinal lymph node (see example 7) at a rate of 200 to 600 pg of peptide per day for seven days, followed by seven days rest.
An initial treatment of 3-8 cycles are conducted.

Example 9 [0137) A fusion protein is made by adding SEQ )D NO. 34 to the 3' end of a nucleotide sequence encoding herpes simplex virus 1 VP22 (SEQ )D NO. 42) in an appropriate mammalian expression vector; the vector used above is suitable. The vector is used to transform HEK 293 cells and 48 to 72 hours later the cells are pelleted, lysed and a soluble extract prepared. The fusion protein is purified by affinity chromatagraphy using an anti-VP22 monoclonal antibody. The purified fusion protein is administered intranodally at a rate of 10 to 100 pg per day for seven days, followed by seven days rest. An initial treatment of 3-8 cycles are conducted.
[0138) Further, the present invention can utilize various aspects of the following: U.S.
Patent Application Nos. 09/380,534, filed on September 1, 1999, entitled A
METHOD OF
INDUCING A CTL RESPONSE; 09/776,232, filed on February 2, 2001, entitled METHOD OF
INDUCING A CTL RESPONSE; 09/715,835, filed on November 16, 2000, entitled AVOmANCE
OF UNDESIRABLE REPLICATION INTERMEDIATES IN PLASMID PROPOGATION;
09/999,186, filed on November 7, 2001, entitled METHODS OF COMMERCIALIZING AN
ANTIGEN; and Provisional U.S. Patent Application No 60/274,063, filed on March 7, 2001, entitled ANTI-NEOVASCULAR VACCINES FOR CANCER.
Table 11 Partial listine of SEO ID NOS.
1 ELAGIGILTV melan-A 26-35 A27L

2 Melan -A rotein Accession number: NP

3 T osinase rotein Accession number: P14679 4 MLLAVLYCLELAGIGILTVYMDGTMSQVGILT pMA2M expression product VILGVLLLIGCWYCRRRNGYRALMDKSLHVG

TQCALTRRCPQEGFDHRDSKVSLQEKNCEPV

MLLAVLYCLELAGIGILTVYMDGTMSQV Liberation or substrate sequence for SEQ m NO. 1 from MA2M

6 MLLAVLYCL t osinase 1-9 7 YMDGTMSQV ' t osinase 369-377 8 EAAGIGILTV melan-A 26-35 cttaagccaccatgttactagctgttttgtactgcctggaactpM~M insert agcagggatcggcatattgacagtgtatatgga tggaacaatgtcccaggtaggaattctgacagtgatcctggga gtcttactgctcatcggctgttggtattgtaga agacgaaatggatacagagccttgatggataaaagtcttcatg ttggcactcaatgtgccttaacaagaagatgcc cacaagaagggtttgatcatcgggacagcaaagtgtctcttca agagaaaaactgtgaacctgtgtagtgagcggc cgc MVLYCLELAGIGILTVYMDGTAVLYCLELAGI Epitope array from pVAXM2 ~ and _77_ GILTVYMDGTMLAVLYCLELAGIGILTVYMD pVAXMI

GTMSLLAVLYCLELAGIGILTV

11 NY-ESO-1 rotein Accession number: P78358 16 AVLYCL t rosinase 4-9 17 MSLLMWITQCKASEKIFYVRCGARGPESRLLE pN157 expression product FYLAMPFATPMEAELARRSLAQDAPPLPVPGV

LLKEFTV SGNILTIRLTAADHRQLQLSISSCLQ

LSLLMWITQCFLPVFLA PPSGQRR

18 MSLLMWITQCKASEKIFYV liberation or substrate sequence for SE ID NO. 12 from N157 19 cttaagccaccatgtccctgttgatgtggatcacgcagtgcaa~ge~ fOrpN157 agcttcggagaaaatcttctacgtacggtgcgg tgccagggggccggagagccgcctgcttgagttctacctcgcc atgcctttcgcgacacccatggaagcagagctg gcccgcaggagcctggcccaggatgccccaccgcttcccgtgc caggggtgcttctgaaggagttcactgtgtccg gcaacatactgactatccgactgactgctgcagaccaccgcca actgcagctctccatcagctcctgtctccagca gctttccctgttgatgtggatcacgcagtgctttctgcccgtg tttttggctcagcctccctcagggcagaggcgc tagtgagaattc 20 MSLLMWITQCKASEKIFYVGLPSIPVHPIGLPSIpBPL expression product PVHPIKASEKIFYVSLLMWITQCKASEKIFYVK

ASEKIFYVRCGARGPESRLLEFYLAMPFATPM

EAELARRSLAQDAPPLPVPGVLLKEFTVSGNIL, TIRL,TAADHRQLQLSISSCLQQLSLLMWITQCF

LPVFLA PPSGQRR

21 atgtccctgttgatgtggatcacgcagtgcaaagcttcggagap$PLInSertCOdlngreglOri aaatcttctatgtgggtcttccaagtattcctg ttcatccaattggtcttccaagtattcctgttcatccaattaa agcttcggagaaaatcttctatgtgtccctgtt gatgtggatcacgcagtgcaaagcttcggagaaaatcttctat gtgaaagcttcggagaaaatcttctacgtacgg tgcggtgccagggggccggagagccgcctgcttgagttctacc tcgccatgcctttcgcgacacccatggaagcag agctggcccgcaggagcctggcccaggatgccccaccgcttcc cgtgccaggggtgcttctgaaggagttcactgt gtccggcaacatactgactatccgactgactgctgcagaccac cgccaactgcagctctccatcagctcctgtctc cagcagctttccctgttgatgtggatcacgcagtgctttctgc ccgtgtttttggctcagcctccctcagggcaga ggcgctagtga 22 IKASEKIFYVSLLMWITQCKASEKIFYVK Substrate in Fig. 6 23 VMTKLGFKV SSX-457_65 24 RQIYVAAFTV PSMA73o-739 25 AQIPEKIQKAFDDIAKYFSKEEWEKMKASEKIFSSX-2 ~ 5_t g3 YVYMKRKYEAMTKLGFKATLPPFMCNKRAE

DFQGNDLDNDPNRGNQVERPQMTFGRLQGIS

PKIMPKKPAEEGNDSEEVPEASGPQNDGKELC

PPGKPTTSEKIHERSGPKRGEHAWTHRLRERK

LVIYEEISDP

_78_ 26 MVMTKLGFKVKASEKIFYVRQIYVAAFTV CTLS1/pCBPexpressionproduct GLPSIPVHPITQCFLPVFLVMTKLGFKVRQIYV

AAFTVKASEKIFYVAQIPEKIQKAFDDIAKYFS

KEEWEKMKASEKIFYVYMKRKYEAMTKLGF

KATLPPFMCNKRAEDFQGNDLDNDPNRGNQ

VERPQMTFGRLQGISPKIMPKKPAEEGNDSEE

VPEASGPQNDGKELCPPGKPTTSEKIHERSGPK

RGEHA W THRLRERKQLV IYEEISDP

27 MAQIPEKIQKAFDDIAKYFSKEEWEKMKASE CTLS2 expression product KIFYVYMKRKYEAMTKLGFKATLPPFMCNKR

AEDFQGNDLDNDPNRGNQVERPQMTFGRLQ

GISPKIMPKKPAEEGNDSEEVPEASGPQNDGK

ELCPPGKPTTSEKIHERSGPKRGEHAWTHRLR

ERKQLVIYEEISDPVMTKLGFKVKASEKIFYV

RQIYVAAFTVGLPSIPVHPITQCFLPVFLVMTK

LGFKVRQIYVAAFTVKASEKIFYV

28 MVMTKLGFKVKASEKIFYVRQIYVAAFTV CTLS3 expression product GLPSIPVHPIAQIPEKIQKAFDDIAKYFSKEEWE

KMKASEKIFYV YMKRKYEAMTKLGFKATLPP

FMCNKRAEDFQGNDLDNDPNRGNQVERPQM

TFGRLQGISPKIMPKKPAEEGNDSEEVPEASGP

QNDGKELCPPGKPTTSEKIHERSGPKRGEHAW

THRLRERK LVIYEEISDP

29 MAQIPEKIQKAFDDIAKYFSKEEWEKMKASE CTLS4 expressipn product KIFYV YMKRKYEAMTKLGFKATLPPFMCNKR

AEDFQGNDLDNDPNRGNQVERPQMTFGRLQ

GISPKIMPKKPAEEGNDSEEVPEASGPQNDGK

ELCPPGKPTTSEKIHERSGPKRGEHAWTHRLR

ERKQLVIYEEISDPTQCFLPVFLVMTKLGFKVR

QIYVAAFTVKASEKIFYV

30 atggtcatgactaaactaggtttcaaggtcaaagcttcggagapCBP Insert COdlrig region aaatcttctatgtgagacagatttatgttgcag ccttcacagtgggtcttccaagtattcctgttcatccaattac gcagtgctttctgcccgtgtttttggtcatgac taaactaggtttcaaggtcagacagatttatgttgcagccttc acagtgaaagcttcggagaaaatcttctacgta gctcaaataccagagaagatccaaaaggccttcgatgatattg ccaaatacttctctaaggaagagtgggaaaaga tgaaagcctcggagaaaatcttctatgtgtatatgaagagaaa gtatgaggctatgactaaactaggtttcaaggc caccctcccacctttcatgtgtaataaacgggccgaagacttc caggggaatgatttggataatgaccctaaccgt gggaatcaggttgaacgtcctcagatgactttcggcaggctcc agggaatctccccgaagatcatgcccaagaagc cagcagaggaaggaaatgattcggaggaagtgccagaagcatc tggcccacaaaatgatgggaaagagctgtgccc cccgggaaaaccaactacctctgagaagattcacgagagatct ggacccaaaaggggggaacatgcctggacccac agactgcgtgagagaaaacagctggtgatttatgaagagatca gcgacccttagtga 31 RQIYVAAFTVKASEKIFYVAQIPEKIQK Fig. 8 substrate/ CTLS1-2 32 FLPWIIRLFL T~2o7-2~5 33 MLLAVLYCLLWSFQTSAFLPWHRLFLMLLAV CTLT2/pMEL expression product LYCLLWSF TSAFLPWHRLFLMLLAVLYCLL

WSFQTSAFLPWHRLFLMLLAVLYCLLWSFQT

SAFLP WHRLFL

34 atgctcctggctgttttgtactgcctgctgtggagtttccagaCTLT2/pMEL insert coding region cctccgcttttctgccttggcatagactcttct tgatgctcctggctgttttgtactgcctgctgtggagtttcca gacctccgcttttctgccttggcatagactctt cttgatgctcctggctgttttgtactgcctgctgtggagtttc cagacctccgcttttctgccttggcatagactc ttcttgatgctcctggctgttttgtactgcctgctgtggagtt tccagacctccgcttttctgccttggcatagac tcttcttgtagtga 35 MELAN-A cDNA Accession number: NM

36 T rosinase eDNA Accession number: NM

37 NY-ESO-1 cDNA Accession number: U87459 38 PSMA rotein Accession number: NP

39 PSMA cDNA Accession number: NM

40 SSX-2 rotein Accession number: NP

41 SSX-2 cDNA Accession number: NM

42 atgacctctcgccgctccgtgaagtcgggtccgcgggaggttccgcgcFrom accession number:

gatgagtacgaggatctgtactacaccccgtcttcaggtatggcgagtccHerpes Simplex virus cgatagtccgcctgacacctcccgccgtggcgccctacagacacgctccoding sequence (VP22) gcgccagaggggcgaggtccgtttcgtccagtacgacgagtcggattat gccctctacgggggctcgtcatccgaagacgacgaacacccggaggt cccccggacgcggcgtcccgtttccggggcggttttgtccggcccggg gcctgcgcgggcgcctccgccacccgctgggtccggaggggccgga cgcacacccaccaccgccccccgggccccccgaacccagcgggtgg cgactaaggcccccgcggccccggcggcggagaccacccgcggca ggaaatcggcccagceagaatccgccgcactcccagacgcccccgcg tcgacggcgccaacccgatccaagacacccgcgcaggggctggcca gaaagctgcactttagcaccgcccccccaaaccccgacgcgccatgga ccccccgggtggccggctttaacaagcgcgtcttctgcgccgcggtcg ggcgcctggcggccatgcatgcccggatggcggcggtccagctctgg gacatgtcgcgtccgcgcacagacgaagacctcaacgaactccttggc atcaccaccatccgcgtgacggtctgcgagggcaaaaacctgcttcag cgcgccaacgagttggtgaatccagacgtggtgcaggacgtcgacgcg gccacggcgactcgagggcgttctgcggcgtcgcgccccaccgageg acctcgagccccagcccgctccgcttctcgccccagacggcccgtega 43 MTSRRSVKSGPREVPRDEYEDLYYTPSSGMAS Accession number: P10233 PDSPPDTSRRGALFTQTRSRQRGEVRFVQYDE Herpes Simplex virus SDYALYGGSSSEDDEHPEVPRTRRPVSGAVLS UL49/VP22 protein sequence GPGPARAPPPFTPAGSGGAGRTPTTAPRAPRT

QRVATKAPAAPAAETTRGRKSAQPESAALPD

APASTAPTFTRSKTPAQGLARKLHFSTAPPNP

DAPWTPRVAGFNKRVFCAAVGRLAAMHARM

AAVQLWDFTMSRPRTDEDLNELLGITTIRVTV

CEGKNLLQRANELVNPDVVQDVDAATATRG

RSAASRFTPTERPRAPARSASRPRRPVE

Melan-A mRNA sequence LOCUS NM 005511 1524 by mRNA PRI 14-OCT-2001 DEFINITION Homo Sapiens melan-A (MLANA), mRNA.

VERSION NM 005511.1 GI:5031912 (SEQ ID NO. 2) /translation="MPREDAHFIYGYPKKGHGHSYTTAEEAAGIGILTVILGVLLLIGCWYCRRRNGY
RALMDKSLHV GTQCALTRRCPQEGFDHRDSKV SLQEKNCEPV VPNAPPAYEKLSAEQSPPP
YSP"
(SEQ ID NO. 35) ORIGIN
1 agcagacaga ggactctcat taaggaaggt gtcctgtgcc ctgaccctac aagatgccaa 61 gagaagatgc tcacttcatc tatggttacc ccaagaaggg gcacggccac tcttacacca 121 cggctgaaga ggccgctggg atcggcatcc tgacagtgat cctgggagtc ttactgctca 181 tcggctgttg gtattgtaga agacgaaatg gatacagagc cttgatggat aaaagtcttc 241 atgttggcac tcaatgtgcc ttaacaagaa gatgcccaca agaagggttt gatcatcggg 301 acagcaaagt gtctcttcaa gagaaaaact gtgaacctgt ggttcccaat gctccacctg 361 cttatgagaa actctctgca gaacagtcac caccacctta ttcaccttaa gagccagcga 421 gacacctgag acatgctgaa attatttctc tcacactttt gcttgaattt aatacagaca 481 tctaatgttc tcctttggaa tggtgtagga aaaatgcaag ccatctctaa taataagtca 541 gtgttaaaat tttagtaggt ccgctagcag tactaatcat gtgaggaaat gatgagaaat 601 attaaattgg gaaaactcca tcaataaatg ttgcaatgca tgatactatc tgtgccagag 661 gtaatgttag taaatccatg gtgttatttt ctgagagaca gaattcaagt gggtattctg 721 gggccatcca atttctcttt acttgaaatt tggctaataa caaactagtc aggttttcga 781 accttgaccg acatgaactg tacacagaat tgttccagta ctatggagtg ctcacaaagg 841 atacttttac aggttaagac aaagggttga ctggcctatt tatctgatca agaacatgtc 901 agcaatgtct ctttgtgctc taaaattcta ttatactaca ataatatatt gtaaagatcc 961 tatagctctt tttttttgag atggagtttc gcttttgttg cccaggctgg agtgcaatgg 1021 cgcgatcttg gctcaccata acctccgcct cccaggttca agcaattctc ctgccttagc 1081 ctcctgagta gctgggatta caggcgtgcg ccactatgcc tgactaattt tgtagtttta 1141 gtagagacgg ggtttctcca tgttggtcag gctggtctca aactcctgac ctcaggtgat 1201 ctgcccgcct cagcctccca aagtgctgga attacaggcg tgagccacca cgcctggctg 1261 gatcctatat cttaggtaag acatataacg cagtctaatt acatttcact tcaaggctca 1321 atgctattct aactaatgac aagtattttc tactaaacca gaaattggta gaaggattta 1381 aataagtaaa agctactatg tactgcctta gtgctgatgc ctgtgtactg ccttaaatgt 1441 acctatggca atttagctct cttgggttcc caaatccctc tcacaagaat gtgcagaaga 1501 aatcataaag gatcagagat tctg Tyrosinase m)tNA sequence LOCUS NM 000372 1964 by mRNA PRI 31-OCT-2000 DEFINITION Homo Sapiens tyrosinase (oculocutaneous albinism IA) (TYR), mRNA.

VERSION NM 000372.1 GI:4507752 (SEQ ID NO. 3) /translation="MLLAVLYCLLWSFQTSAGHFPRACV SSKNLMEKECCPPW SGDRS
PCGQLSGRGSCQNIL,LSNAPLGPQFPFTGVDDRESWPSVFYNRTCQCSGNFMGFNCGNCKFG
FWGPNCTERRLLVRRNIFDLSAPEKDKFFAYLTLAKHTISSDYVIPIGTYGQMKNGSTPMFND
INIYDLFV WMHYYV SMDALLGGSEIWRD>DFAHEAPAFLPWHRLFLLRWEQEIQKLTGDENF
TIPYWDWRDAEKCDICTDEYMGGQHPTNPNLLSPASFFSSWQIVCSRLEEYNSHQSLCNGTP
EGPLRRNPGNHDKSRTPRLPSSADVEFCLSLTQYESGSMDKAANFSFRNTLEGFASPLTGIAD
ASQSSMHNALHIYMNGTMSQVQGSANDPIFLLHHAFVDSIFEQWLRRHRPLQEVYPEANAPI
GHNRESYMVPFIPLYRNGDFFISSKDLGYDYSYLQDSDPDSFQDYIKSYLEQASRIWSWLLGA
AMVGAVLTALLAGLVSLLCRHKRKQLP EEKQPLLMEKEDYHSLYQSHL"
(SEQ ID NO. 36) ORIGIN
1 atcactgtag tagtagctgg aaagagaaat ctgtgactcc aattagccag ttcctgcaga 61 ccttgtgagg actagaggaa gaatgctcct ggctgttttg tactgcctgc tgtggagttt 121 ccagacctcc gctggccatt tccctagagc ctgtgtctcc tctaagaacc tgatggagaa 181 ggaatgctgt ccaccgtgga gcggggacag gagtccctgt ggccagcttt caggcagagg 241 ttcctgtcag aatatccttc tgtccaatgc accacttggg cctcaatttc ccttcacagg 301 ggtggatgac cgggagtcgt ggccttccgt cttttataat aggacctgcc agtgctctgg 361 caacttcatg ggattcaact gtggaaactg caagtttggc ttttggggac caaactgcac 421 agagagacga ctcttggtga gaagaaacat cttcgatttg agtgccccag agaaggacaa 481 attttttgcc tacctcactt tagcaaagca taccatcagc tcagactatg tcatccccat 541 agggacctat ggccaaatga aaaatggatc aacacccatg tttaacgaca tcaatattta 601 tgacctcttt gtctggatgc attattatgt gtcaatggat gcactgcttg ggggatctga 661 aatctggaga gacattgatt ttgcccatga agcaccagct tttctgcctt ggcatagact 721 cttcttgttg cggtgggaac aagaaatcca gaagctgaca ggagatgaaa acttcactat 781 tccatattgg gactggcggg atgcagaaaa gtgtgacatt tgcacagatg agtacatggg 841 aggtcagcac cccacaaatc ctaacttact cagcccagca tcattcttct cctcttggca 901 gattgtctgt agccgattgg aggagtacaa cagccatcag tctttatgca atggaacgcc 961 cgagggacct ttacggcgta atcctggaaa ccatgacaaa tccagaaccc caaggctccc 1021 ctcttcagct gatgtagaat tttgcctgag tttgacccaa tatgaatctg gttccatgga 1081 taaagctgcc aatttcagct ttagaaatac actggaagga tttgctagtc cacttactgg 1141 gatagcggat gcctctcaaa gcagcatgca caatgccttg cacatctata tgaatggaac 1201 aatgtcccag gtacagggat ctgccaacga tcctatcttc cttcttcacc atgcatttgt 1261 tgacagtatt tttgagcagt ggctccgaag gcaccgtcct cttcaagaag tttatccaga 1321 agccaatgca cccattggac ataaccggga atcctacatg gttcctttta taccactgta 1381 cagaaatggt gatttcttta tttcatccaa agatctgggc tatgactata gctatctaca 1441 agattcagac ccagactctt ttcaagacta cattaagtcc tatttggaac aagcgagtcg 1501 gatctggtca tggctccttg gggcggcgat ggtaggggcc gtcctcactg ccctgctggc 1561 agggcttgtg agcttgctgt gtcgtcacaa gagaaagcag cttcctgaag aaaagcagcc 1621 actcctcatg gagaaagagg attaccacag cttgtatcag agccatttat aaaaggctta 1681 ggcaatagag tagggccaaa aagcctgacc tcactctaac tcaaagtaat gtccaggttc 1741 ccagagaata tctgctggta tttttctgta aagaccattt gcaaaattgt aacctaatac 1801 aaagtgtagc cttcttccaa ctcaggtaga acacacctgt ctttgtcttg ctgttttcac 1861 tcagcccttt taacattttc ccctaagccc atatgtctaa ggaaaggatg ctatttggta 1921 atgaggaact gttatttgta tgtgaattaa agtgctctta tttt NY-ESO-1 mRNA sequence LOCUS HSU87459 752 by mRNA PRI 22-DEC-1999 DEFINITION Human autoimmunogenic cancer/testis antigen NY-ESO-1 mRNA, complete cds.

VERSION U87459.1 GI:1890098 (SEQ ID NO. 11) /translation="MQAEGRGTGGSTGDADGPGGPGII'DGPGGNAGGPGEAGATGGRGPRGAGAAR
ASGPGGGAPRGPHGGAASGLNGCCRCGARGPESRLLEFYLAMPFATPMEAELARRSLAQDA
PPLPVPGVLLKEFTVSGNILTIRLTAADHRQLQLSISSCLQQLSLLM
WITQCFLPVFLAQPPSGQRR"
(SEQ ID NO. 37) ORIGIN
1 atcctcgtgg gccctgacct tctctctgag agccgggcag aggctccgga gccatgcagg 61 ccgaaggccg gggcacaggg ggttcgacgg gcgatgctga tggcccagga ggccctggca 121 ttcctgatgg cccagggggc aatgctggcg gcccaggaga ggcgggtgcc acgggcggca 181 gaggtccccg gggcgcaggg gcagcaaggg cctcggggcc gggaggaggc gccccgcggg 241 gtccgcatgg cggcgcggct tcagggctga atggatgctg cagatgcggg gccagggggc 301 cggagagccg cctgcttgag ttctacctcg ccatgccttt cgcgacaccc atggaagcag 361 agctggcccg caggagcctg gcccaggatg ccccaccgct tcccgtgcca ggggtgcttc 421 tgaaggagtt cactgtgtcc ggcaacatac tgactatccg actgactgct gcagaccacc 481 gccaactgca gctctccatc agctcctgtc tccagcagct ttccctgttg atgtggatca 541 cgcagtgctt tctgcccgtg tttttggctc agcctccctc agggcagagg cgctaagccc 601 agcctggcgc cccttcctag gtcatgcctc ctcccctagg gaatggtccc agcacgagtg 661 gccagttcat tgtgggggcc tgattgtttg tcgctggagg aggacggctt acatgtttgt 721 ttctgtagaa aataaaactg agctacgaaa as PSMA cDNA sequence LOCUS NM 004476 2653 by mRNA PRI O1-NOV-2000 DEFINITION Homo Sapiens folate hydrolase (prostate-specific membrane antigen) 1 (FOLH 1 ), mRNA.

VERSION NM 004476.1 GI:4758397 (SEQ 1D NO. 38) /translation="MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNIT
PKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYD
VLLSYPNKTHPNYISIlNEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNY
ARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVK
SYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDA
QKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRG
AVEPDRYVILGGH RDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEF
GLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGF
EGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPL
YHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIY
SISMKHPQEMKTYSV SFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAF
IDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFT
VQAAAETLSEVA"
(SEQ ID NO. 39) ORIGIN
1 ctcaaaaggg gccggatttc cttctcctgg aggcagatgt tgcctctctc tctcgctcgg 61 attggttcag tgcactctag aaacactgct gtggtggaga aactggaccc caggtctgga 121 gcgaattcca gcctgcaggg ctgataagcg aggcattagt gagattgaga gagactttac 181 cccgccgtgg tggttggagg gcgcgcagta gagcagcagc acaggcgcgg gtcccgggag 241 gccggctctg ctcgcgccga gatgtggaat ctccttcacg aaaccgactc ggctgtggcc 301 accgcgcgcc gcccgcgctg gctgtgcgct ggggcgctgg tgctggcggg tggcttcttt 361 ctcctcggct tcctcttcgg gtggtttata aaatcctcca atgaagctac taacattact 421 ccaaagcata atatgaaagc atttttggat gaattgaaag ctgagaacat caagaagttc 481 ttatataatt ttacacagat accacattta gcaggaacag aacaaaactt tcagcttgca 541 aagcaaattc aatcccagtg gaaagaattt ggcctggatt ctgttgagct agcacattat 601 gatgtcctgt tgtcctaccc aaataagact catcccaact acatctcaat aattaatgaa 661 gatggaaatg agattttcaa cacatcatta tttgaaccac ctcctccagg atatgaaaat 721 gtttcggata ttgtaccacc tttcagtgct ttctctcctc aaggaatgcc agagggcgat 781 ctagtgtatg ttaactatgc acgaactgaa gacttcttta aattggaacg ggacatgaaa 841 atcaattgct ctgggaaaat tgtaattgcc agatatggga aagttttcag aggaaataag 901 gttaaaaatg cccagctggc aggggccaaa ggagtcattc tctactccga ccctgctgac 961 tactttgctc ctggggtgaa gtcctatcca gatggttgga atcttcctgg aggtggtgtc 1021 cagcgtggaa atatcctaaa tctgaatggt gcaggagacc ctctcacacc aggttaccca 1081 gcaaatgaat atgcttatag gcgtggaatt gcagaggctg ttggtcttcc aagtattcct 1141 gttcatccaa ttggatacta tgatgcacag aagctcctag aaaaaatggg tggctcagca 1201 ccaccagata gcagctggag aggaagtctc aaagtgccct acaatgttgg acctggcttt 1261 actggaaact tttctacaca aaaagtcaag atgcacatcc actctaccaa tgaagtgaca 1321 agaatttaca atgtgatagg tactctcaga ggagcagtgg aaccagacag atatgtcatt 1381 ctgggaggtc accgggactc atgggtgttt ggtggtattg accctcagag tggagcagct 1441 gttgttcatg aaattgtgag gagctttgga acactgaaaa aggaagggtg gagacctaga 1501 agaacaattt tgtttgcaag ctgggatgca gaagaatttg gtcttcttgg ttctactgag 1561 tgggcagagg agaattcaag actccttcaa gagcgtggcg tggcttatat taatgctgac 1621 tcatctatag aaggaaacta cactctgaga gttgattgta caccgctgat gtacagcttg 1681 gtacacaacc taacaaaaga gctgaaaagc cctgatgaag gctttgaagg caaatctctt 1741 tatgaaagtt ggactaaaaa aagtccttcc ccagagttca gtggcatgcc caggataagc 1801 aaattgggat ctggaaatga ttttgaggtg ttcttccaac gacttggaat tgcttcaggc 1861 agagcacggt atactaaaaa ttgggaaaca aacaaattca gcggctatcc actgtatcac 1921 agtgtctatg aaacatatga gttggtggaa aagttttatg atccaatgtt taaatatcac 1981 ctcactgtgg cccaggttcg aggagggatg gtgtttgagc tagccaattc catagtgctc 2041 ccttttgatt gtcgagatta tgctgtagtt ttaagaaagt atgctgacaa aatctacagt 2101 atttctatga aacatccaca ggaaatgaag acatacagtg tatcatttga ttcacttttt 2161 tctgcagtaa agaattttac agaaattgct tccaagttca gtgagagact ccaggacttt 2221 gacaaaagca acccaatagt attaagaatg atgaatgatc aactcatgtt tctggaaaga 2281 gcatttattg atccattagg gttaccagac aggccttttt ataggcatgt catctatgct 2341 ccaagcagcc acaacaagta tgcaggggag tcattcccag gaatttatga tgctctgttt 2401 gatattgaaa gcaaagtgga cccttccaag gcctggggag aagtgaagag acagatttat 2461 gttgcagcct tcacagtgca ggcagctgca gagactttga gtgaagtagc ctaagaggat 2521 tctttagaga atccgtattg aatttgtgtg gtatgtcact cagaaagaat cgtaatgggt 2581 atattgataa attttaaaat tggtatattt gaaataaagt tgaatattat atataaaaaa 2641 aaaaaaaaaa aaa NM 003147 Homo Sapiens synovial sarcoma, X breakpoint 2 (SSX2), ml2NA
LOCUS NM-003147 766 by mRNA PRI 14-MAR-2001 DEFINITION Homo Sapiens synovial sarcoma, X breakpoint 2 (SSX2), mRNA.
ACCESSION NM_003147 VERSION NM 003147.1 GI:10337582 SEQ ID NO. 40 /translation=~~MNGDDAFARRPTVGAQIPEKIQKAFDDIAKYFSKEEWEKMKASE
KIFYVYMKRKYEAMTKLGFKATLPPFMCNKRAEDFQGNDLDNDPNRGNQVERPQMTFG
RLQGISPKIMPKKPAEEGNDSEEVPEASGPQNDGKELCPPGKPTTSEKIHERSGPKRG
EHAWTHRLRERKQLVIYEEISDPEEDDE"

1 ctctctttcg attcttccat actcagagta cgcacggtct gattttctct ttggattctt 61 ccaaaatcag agtcagactg ctcccggtgc catgaacgga gacgacgcct ttgcaaggag 121 acccacggtt ggtgctcaaa taccagagaa gatccaaaag gccttcgatg atattgccaa 181 atacttctct aaggaagagt gggaaaagat gaaagcctcg gagaaaatct tctatgtgta 241 tatgaagaga aagtatgagg ctatgactaa actaggtttc aaggccaccc tcccaccttt 301 catgtgtaat aaacgggccg aagacttcca ggggaatgat ttggataatg accctaaccg 361 tgggaatcag gttgaacgtc ctcagatgac tttcggcagg ctccagggaa tctccccgaa 421 gatcatgccc aagaagccag cagaggaagg aaatgattcg gaggaagtgc cagaagcatc 481 tggcccacaa aatgatggga aagagctgtg ccccccggga aaaccaacta cctctgagaa 541 gattcacgag agatctggac ccaaaagggg ggaacatgcc tggacccaca gactgcgtga 601 gagaaaacag ctggtgattt atgaagagat cagcgaccct gaggaagatg acgagtaact 661 cccctcaggg atacgacaca tgcccatgat gagaagcaga acgtggtgac ctttcacgaa 721 catgggcatg gctgcggacc cctcgtcatc aggtgcatag caagtg

Claims (21)

WHAT IS CLAIMED IS:

1. A method of identifying a polypeptide suitable for epitope liberation, the method comprising the steps of:
identifying an epitope of interest;
providing a substrate polypeptide sequence comprising the epitope, wherein the substrate polypeptide permits processing by a proteasome;
contacting the substrate polypeptide with a composition comprising the proteasome, under conditions that support processing of the substrate polypeptide by the proteasome; and assaying for liberation of the epitope.

2. The method of claim 1, wherein the epitope is embedded in the substrate polypeptide.

3. The method of claim 1, wherein the epitope is a housekeeping epitope.

4. The method of claim 1, wherein the substrate polypeptide is a synthetic peptide.

5. The method of claim 1, wherein the substrate polypeptide is a fusion protein.

6. The method of claim 1, wherein the contacting step comprises immunization with the substrate polypeptide.

7. The method of claim 1, wherein the substrate polypeptide is encoded by a polynucleotide.

8. The method of claim 7, wherein the contacting step comprises immunization with a vector comprising the polynucleotide.

9. The method of claim 7, wherein the contacting step comprises transforming a cell with a vector comprising the polynucleotide.

10. The method of claim 1, wherein the proteasome processing takes place in vitro.

11. The method of claim 1, wherein the assaying step consists of a technique selected from the group consisting of mass spectrometry, N-terminal pool sequencing, and HPLC.

12. The method of claim 1, wherein the assaying step comprises a T cell target recognition assay.

13. The method of claim 1, wherein the substrate polypeptide further comprises an array of additional epitopes.

14. The method of claim 13, wherein the array comprises a housekeeping and an immune epitope.

15. The method of claim 1, wherein the substrate polypeptide further comprises an array of epitopes and epitope clusters.

16. The method of Claim 1, wherein the proteasome is an immune proteasome.

17. A vector comprising a housekeeping epitope expression cassette, wherein the housekeeping epitope is derived from a target-associated antigen, and wherein the housekeeping epitope is liberatable from a translation product of the cassette by immunoproteasome processing.

18. The vector of claim 17, wherein the expression cassette encodes an array of two or more epitopes or at least one epitope and at least one epitope cluster.

19. The vector of claim 17, wherein the target-associated antigen is an antigen derived from or associated with a tumor or an intracellular parasite.

20. A method of activating a T cell comprising contacting the vector of claim 17 with an APC and contacting said APC with a T cell.

21. A substrate polypeptide comprising a housekeeping epitope wherein the housekeeping epitope can be liberated by immunoproteasome processing in a pAPC.